THE USE OF WT1-QPCR TO MEASURE AND DETECT MINIMAL RESIDUAL DISEASE IN ACUTE MYELOID LEUKEMIA. Ava Greco

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1 THE USE OF WT1-QPCR TO MEASURE AND DETECT MINIMAL RESIDUAL DISEASE IN ACUTE MYELOID LEUKEMIA Ava Greco

2 REVIEW OF LITERATURE What is Leukemia? Abnormal growth of cells in the blood stream Progresses rapidly without treatment DNA is damaged in normal stems cells leading to leukemic blasts (Applebaum et al., 2003) Leukemic cells look like normal immature blood cells, but they have genetic changes. Symptoms do not occur until 1 trillion of leukemia cells About 3% of your blood ved=0ahukewj3hiab7nlyahvb04mkhuxcarmqjrwibw&url=http%3a%2f%2fslideplayer.com%2 Fslide%2F %2F&psig=AOvVaw1AgfwHePC5l1KGg5Y6J1eh&ust=

3 REVIEW OF LITERATURE Leukemia Acute myeloid leukemia (AML) Chronic myeloid leukemia (CML) Acute lymphocytic leukemia (ALL) Chronic lymphocytic leukemia (CLL) Acute vs. chronic Different growth rate

4 REVIEW OF LITERATURE Diagnosis Blood tests Complete blood count (CBC) Bone marrow biopsy Immature white blood cells At least 20% blasts to be leukemia Pre-leukemia state= Myelodysplasia (lls.org)

5 TREATMENT/RESPONSE Treatment includes chemotherapy or stem cell transplant Morphologic leukemia-free state: <5% blasts in the bone marrow (1 in 20 cells) Normal blood counts (Cheson et al. 2003)

6 REVIEW OF LITERATURE What is Minimal Residual Disease (MRD)? Leukemic cells remaining during or after treatment Major cause of relapse in leukemia Gomez. A

7 What are the current ways to detect residual leukemia cells Morphology Cytogenetics Flow cytometry qpcr

8 REVIEW OF LITERATURE qpcr Polymerase chain reaction Amplifies a single copy or a few copies of a segment of DNA 1. DNA is separated into single strands 2. Primers and polymerase bind 3. Allows nucleotides (polymers) to attach and synthesize DNA Data collected is analyzed by PCR software

9 REVIEW OF LITERATURE

10 RESEARCH GAP An effective way to detect residual leukemia cells to better stratify if the patient will relapse or will go into remission, and improve treatment strategies to target them

11 RESEARCH QUESTION Can WT1 qpcr detect MRD more efficiently than the other means of detecting MRD?

12 WT1 Wilms tumor gene Overexpressed in leukemia Represents a reliable marker Provides a sensitive method for monitoring the disease after treatment An increase level of WT1 during follow ups of AML patients is always indicative of relapse (Cilloni et al. 2006)

13 HYPOTHESIS H: The use of qpcr to detect WT1 overexpression will be able to detect MRD with at least 1/1000 cells and at most 1/100,000 cells. H0: qpcr to detect WT1 will not be able to detect MRD.

14 METHODS 1. Design the assay 2. Use qpcr 3. Crate a standard curve

15 METHODS 1. Dilutions of cells HL60 (WT1) and MNC (ABL) 2. Extract RNA 3. Make cdna to do qpcr

16 METHODS PCR reaction mix component Volume/20 µl (single reaction) TaqMan Gene Expression Assay 1.0 TaqMan Gene Expression Master Mix 10.0 cdna template 4.0 RNase-free water 5.0 1/10 ABL 1/100 ABL 1/1,000 ABL 1/10,000 ABL 1/100,000 ABL NTC ABL HL60 MNC 1/10 WT1 1/100 WT1 1/1,000 WT1 1/10,000 WT1 1/100,000 WT1 NTC WT1 HL60 MNC

17 RESULTS Undiluted All Threshold cycle (Ct) value is the intersection between an amplification curve and a threshold line. It is a relative measure of the concentration of target in the PCR reaction 1/100,000

18 RESULTS

19 RESULTS It is reliable for at least 1/1000 Translates to a sensitivity of 10-4

20 FUTURE STEPS We are going to re-perform the standard curve Test in our samples from patients to compare to flow cytometry and see which is better to detect

21 ACKNOWLEDGEMENTS Alexandra Gomez, M.D. Weill Cornell Medical College /New York Presbyterian Hospital Mrs. Carnahan, Mrs. O Brien, Mr. Inglis, and Ms. Dyer Family

22 BIBLIOGRAPHY Bergmann, Lothar, et al. High Levels of Wilms' Tumor Gene (wt1) mrna in Acute Myeloid With a Worse Long-Term Outcome. Blood Journal. Leukemias Are Associated Cheson, B. D.... Bloomfield, C. D. (2003). Revised recommendations of the International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia. JCO, 21(24), Cilloni, Daniela, et al. Real-Time Quantitative Polymerase Chain Reaction Detection of Minimal Residual Disease by Standardized WT1 Assay to Enhance Risk Stratification in Acute Myeloid Leukemia: A European LeukemiaNet Study. Journal of Clinical Oncology. Gknation. Acute Myeloid Leukemia. Leukemia & Lymphoma Society, 26 Feb. 2015, myeloid-leukemia. Anonymous. Diagnosis. Leukemia & Lymphoma Society, 26 Feb. 2015, leukemia/diagnosis. Iland, Harry, et al. Myeloid Leukemia: Methods and Protocols. Humana Press Inc., Inoue, Kazushi, et al. WT1 as a New Prognostic Factor and a New Marker for the Detection of Minimal Residual Disease in Acute Leukemia. Blood Journal. Inoue, Kazushi, et al. Long-Term Follow-Up of Minimal Residual Disease in Leukemia Tumor Gene) Expression Levels. Blood Journal. Patients by Monitoring WT1 (Wlims Inoue, Kazushi, et al. Aberrant Overexpression of the Wilms Tumor Gene (WT1) in Human Leukemia. Blood Journal. Technologies Bio-Rad, What is Real-Time PCR (QPCR)? What is Real-Time PCR (QPCR)? Applications & rad.com/en-us/applications-technologies/what-real- time-pcr-qpcr.