The Paul O'Gorman Leukemia Research Centre is focused on understanding hematopoietic stem cells and their role in leukemia development. The center aims to identify new therapeutic targets for leukemia by exploring CML stem/progenitor cells. The document outlines a 10-week project to produce an RNA bank from CML cell lines and patient samples and characterize the samples through colony formation assays, PCR to determine BCR-ABL breakpoints, flow cytometry for CD34/CD38 expression, and FISH to visualize the fusion oncogene. Preliminary results are shown for several patient samples that were analyzed.
Mutations in Chronic myeloid leukaemia and Imatinib resistanceDr Sandeep Kumar
some corrections over previous presentation on CML. Covers topics like - pathophysiology of CML, Mutations discussed in detail, TKI resistance in various mutations and treatment options. Also Imatinib resistance has been discussed in detail.
Introduction
Oncogenes and Tumor Suppressor Genes
Overexpression of cyclin D1
Loss of p16 Function
Loss of signalling Contributes to abnormal cell proliferation and malignancy
Summary
Questions
Mutations in Chronic myeloid leukaemia and Imatinib resistanceDr Sandeep Kumar
some corrections over previous presentation on CML. Covers topics like - pathophysiology of CML, Mutations discussed in detail, TKI resistance in various mutations and treatment options. Also Imatinib resistance has been discussed in detail.
Introduction
Oncogenes and Tumor Suppressor Genes
Overexpression of cyclin D1
Loss of p16 Function
Loss of signalling Contributes to abnormal cell proliferation and malignancy
Summary
Questions
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Concurrent Determination of ABO RhD Blood Types and the HIV-1 Resistance Mark...Thermo Fisher Scientific
For optimal outcomes when matching bone marrow donors with their recipients, it is preferable to use bone marrow of identical or compatible blood types. Bone marrow registries thus require high resolution HLA genotyping data to match donor specimens with their recipients. We developed a research assay to aid in these investigations, which utilizes buccal swab DNA from potential donors to determine the ABO and Rh-antigen genotypes. In addition, the assay detects a 32 bp deletion in the CCR5 gene. Homozygous carriers of this deletion are resistant to HIV-1 infection, and thus could be valuable stem cell donors for HIV-infected recipients
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MilliporeSigma's Toni Steiner recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating how culture conditions can influence drug transporter expression and activity in renal proximal tubule epithelial cells.
This presentation consists of topics related to oncogene, proto oncogene, Tumor suppresor gene, Ras gene family and structure and functions of tumor suppressor gene.
Derivation of highly enriched cultures of differentiated cells from human par...Nikolay Turovets
California Institute for Regenerative Medicine (CIRM) & Medical Research Council (MRC)
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14 June, 2010, San Francisco, CA
Workshop report: http://www.cirm.ca.gov/files/PDFs/Publications/Human_SCNT_Workshop_Report.pdf
Downloadable slide decks are a great tool for self study and teaching purposes. They are non-certified resources available to enhance your knowledge.
Review a downloadable slide deck by Michael W. N. Deininger, MD, PhD, covering the most clinically relevant new data reported from Community Oncology Clinical Debates: Chronic Myelogenous Leukemia.
Target Audience
This educational activity has been designed to meet the unique learning needs of hematologists, medical oncologists, oncology fellows, and pathologists involved in the treatment of patients with chronic myelogenous leukemia (CML).
This program is designed to provide hematologists/oncologists with the latest clinical updates on the treatment of patients with CML in the frontline and relapsed settings. Key elements of this program will highlight many of the clinical challenges faced by physicians as they select appropriate therapeutic regimens for their patients. Assisting clinicians in gaining further perspective regarding the importance of response criteria, clinical monitoring of treatment failure/suboptimal response/disease resistance, timing for switching therapy to obtain maximum therapeutic benefit, the significance of BCR-ABL mutational analysis in patients and how this can assist in selecting therapy and designing individualized approaches to managing CML will be important discussion points throughout the symposium.
Get to Know About the Molecular Cytogenetic Analysis for Acute Lymphoblastic Leukemia. This test will diagnose and monitor the treatment response of patients.
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Molecular Diagnostics
1. BCR-ABL1 Splice Patterns &
Generation of CML RNA bank
Placement Presentation
Deborah Reilly
7th May 2015
2. Paul O’Gorman Leukemia Research Centre
• Is a division of the University of Glasgow's Institute of Cancer Sciences
• Situated in the grounds of Gartnavel General Hospital which is located in the west end of
Glasgow
• Research and objectives:
- Centre’s research is focused around the understanding of the haematopoietic stem
cell (HSC)and its key role in the development of the vast variety of leukemic diseases
- Centre’s objective is to explore and identify therapeutic targets for leukaemia and in
turn produce new medication for patients
3. The Philadelphia chromosome, hallmark of CML,
results from translocation between chromosomes 9 and 22
Nature 1973, 243, 290 - 293
Bcr-Abl
# 9 # 22
c-Abl
Bcr
Ph1
Janet RowleyPeter Nowell
David Hungerford
Science 1960,132:1497
5. Project Outline (10 weeks)
• Set up/Learn techniques (2 weeks).
• Produce a bank of RNA/cDNA from:
(i) peripheral blood samples,
(ii) CML cell lines, and
(iii) primary CD34+ CML cells
• Determine baseline CFC
• Determine the BCR-ABL1 breakpoint using PCR
• Determine percentage CD34+CD38- by flow cytometry
• Use FISH to visualise the fusion oncogene
6. Functional Analysis of CML Stem/Progenitor Cells
Colony Forming Cells (CFC) assay
- Measures relatively mature, committed progenitor cells
CD34+ cells
Methylcellulose (H4034)
12-14 days
in culture
Total number of
colonies counted
8. PCR Changes
• The dNTPs dilution concentration was noticed to be x10 out – was amended
• The annealing temperature for the BCRABL1 F/R primers was calculated >60°C
The annealing temperature for the Actin 1/2 primers was calculated to be 58°C
- was running at 55°C , so had to be increased for BCRABL1
• Fresh cell line source as positive control
• An appropriate PCR programme on another thermal cycler was chosen
• Optimum PCR conditions discovered:
- For the BCRABL1 F/R primers:
- 3µM [Mg2+] addition with an annealing temperature of 65°C
- For the Actin 1/2 primers:
- 2µM [Mg2+] addition with an annealing temperature of 60°C
9. PCR Results
KY01
Cell lineages at their optimum PCR for primers BCRABL1 F/R and Actin 1/2:
b3a2 (13)
b2a2 (14)
Em2 Bv173 K562
KY01 NB4 Em2 Bv173
KY01
Splice variants of primary CD34+ CML samples:
KCL22 K562 CML 378
KY01 Em2 K562 CML 342
Were not
carried out in
the optimum
PCR conditions
Actin (housekeeping control)
10. Flow cytometry Results
• All of the primary samples were accessed for their viability and CD34+ CD38- expression using
flow cytometry
CML 404:
Q4 = 12.4%
11. FISH
• Probe properties:
- BCR = Green
- ABL = Red
- Fusion = Yellow
• Cell lineage K562 was used as the positive control:
- Larger than CD34+ stem cells
- Number of BCR:ABL signals roughly 3:2
- Have around 10 copies of the fusion oncogene per cell
• Primary CD34+ CML 416 sample:
- 1 BCR signal, 1 ABL signal and 2 fusion signals per cell
K562 CML416
In 1960 David Hungerford and Peter C. Nowell provided the first evidence for a genetic link to cancer when they identified an abnormally small chromosome in cells from patients with CML. They both worked in Philadelphia and therefore called this small chromosome the Philadelphia chromosome. In 1973, Janet Rowley saw, using Chromosomal banding techniques, that the Ph chromosome resulted from a reciprocal translocation between chromosome 9 and 22, and in the 1980s, the Ph chromosome was shown to carry a unique fusion gene, termed BCR-ABL, which is now believed to be the principal cause of CML.