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Translational Research Bridging the Gap

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29 diapositives · 633 Views · Dec 01, 2014

Translational Research Bridging the Gap. Asim Belgaumi King Faisal Specialist Hospital & Research Centre Riyadh Saudi Arabia. Research Scientist. Clinical Researcher. Research Interest Concept Development Methodology Sample Choice Result Interpretation Practical Applicability.

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  1. Translational ResearchBridging the Gap Asim Belgaumi King Faisal Specialist Hospital & Research Centre Riyadh Saudi Arabia

  2. Research Scientist Clinical Researcher Research Interest Concept Development Methodology Sample Choice Result Interpretation Practical Applicability

  3. Basic Molecular Biology • Understanding disease pathophysiology • Molecular diagnostics • Molecularly directed therapy

  4. Concepts of Translational Research • Interaction between the clinician and the scientist • Questions from the clinical setting are brought for discussion for potential answers • Potential results and ideas are brought from the laboratory for clinical relevance • Early Translation of the laboratory findings to clinical application

  5. Bench Bedside

  6. The Clinician Scientist • A clinician actively involved in patient care • Research background • Understands laboratory research methodologies • Active interaction with the Scientists

  7. Translational Studies in Pediatric Leukemia

  8. B-ALLs frequently carry non-random chromosomal translocations. • More than 50 distinct rearrangements have been described. • However, only four, i.e. t(12;21), t(1;19), t(9;22) and t(4;11), are frequently observed and constitute about 40% of cases. • Lead to the formation of fusion genes relevant in leukemogenesis. • These translocations have prognostic significance.

  9. Chromosomal translocations • Routine Karyotyping • Time and sample consuming • Inefficient • Can be detected by FISH and Southern blot • Time and sample consuming • Directed towards one rearrangement at a time • Not appropriate for quantification • Can be detected by RT-PCR • Can detect multiple rearrangements in one tube • Not appropriate for quantification

  10. Aims • Develop a quantitative real-time MPX-RT-PCR for rapid and reliable detection of common ALL-specific translocations. • Define the distribution of translocations in childhood B-ALL in non-Western populations.

  11. Monochrome Real Time Multiplex RT-PCR MLL-AF4 BM or PB -------------cDNA E2A-PBX1 E2A/PBX TEL/AML1 MLL/AF4 primers TEL-AML1

  12. MULTIPLEX REAL TIME RT-PCR Each amplicon has a specific melting peak MLL-AF4E2A-PBX1 83.6+/-0.3oC93.9+/-0.4oC TEL-AML1 86+/-0.5oC

  13. A Each patient sample yields a peak Patient’s samples with translocations RS4;11 Patient’s samples without translocations showing HPRT amplification 697 REH

  14. t(9;22) was detected in a tandem reaction SupB15 Positive patients

  15. Distribution of Translocations in ALL from India 12;21 9;22 1;19 4;11 none 18 (7%) 14 (5%) 18 (7%) 0 209 N=259

  16. Comparison of the distribution of translocations in different world regions %12;21 9;22 1;19 4;11 UK 19 1.5 2.5 1.1 USA 22 2.2 3.8 1.2 India 7 5 7 none p<0.005 p<0.005 p<0.005

  17. CONCLUSIONS • Real time MPX RT-PCR can be used for rapid, simple and reliable classification of pediatric ALL. • Molecular subgroups of ALL are differently distributed in different populations. • Indian patients showed an increase in BCR-ABL and a decrease in TEL-AML. • Saudi patients had a TEL-AML frequency similar to Western populations and lower BCR-ABL. • These factors may be critical in clinical outcome and consequently, in the design of new therapies.

  18. OBJECTIVES OF THE STUDY • Development of Real-Time PCR assay for • Identification and Quantification • Species:Candida (albicans, krusei, tropicalis) • Aspergillus (flavus, fumigatus) • Specific primers for each species (rRNA gene) • Probe free • Duplex PCR • Validation on proven fungal positive samples

  19. Methods Extraction Culture Species Genomic DNA PCR PCR Cloning Analysis for Specificity, Tm and Sensitivity Extraction pCR 2.1-TOPO Standard DNA E. coli Analysis for Sensitivity and to make Standard Curve

  20. Table 1 Characteristics of Real Time PCR for Each Species 1 fg  200 plasmid copies  1.7-2 cells

  21. Table 2: Specificity of Real Time PCR A total of 60 isolates, including Candida, Aspergillus, Cryptococcus, Tricosporon and Alternaria, were used.

  22. Patient 1 Quantification Report: Candida albicansandAspergillus favus 305 C. albicans cells/ml blood Standard positive control Patient Sample Candida tropicalisandCandida krusei Patient 2 Quantification Report: 3154 C. krusei cells/ml blood Standard positive control Patient Sample Patient 2 Quantification Report Aspergillus fumigatus 427 A. flavus cells/mm3 Standard positive control Patient Sample

  23. CONCLUSIONS • Probe-free, real time PCR assays were successful in • identification of • C. albicans, C. krusei, C. tropicalis, A. flavus and A. fumigatus. • Probe-free real-time PCR allows reliable quantification of • fungal load in patient • Sensitivity:as low as40cells / ml of blood • forC. albicans, C. krusei, C. tropicalis, A. flavus • 4 cell/ml of blood for A. fumigatus. • ( 1ml PB 20 ul DNA: 1ul for Real Time PCR)

  24. Characteristic of the techniques currently employed for MRD detection in ALL FCM PCR/CT PCR/Ig or TCR PCR/TdT Sensitivity10-3-- 10-4 10-4-- 10-6 10-4-- 10-5 Applicability 95% all types of ALL pre-B-ALL60-98% 30-40% 90-95% (except B-ALL) T-ALL90-95% 15-35% 90-95% 20% of AML Advantages applicable easy & cheap applicability if + easy & cheap for most Pts sensitive & sensitive & sensitive relative cheap leuk-specific pts-specific 1-2 days2-3days 2-3 days 2-3 days Dis- limited-sensitivity minority of pts Time-consuming at dig. Only PB and CSF Advantages need for preferably PCR contamin- need for preferably two No leukemia aberrant Immuno- ation PCR targets or pt specific phenotype per patient normal BM TdT + BM TdT + after BMT and therapy

  25. Quantification of TdT expression in normal peripheral blood 110-4 • Normal PB (N: 24) • Range 0 - 0.007 • Mean 0.0006 • Median 0

  26. Do TdT positive cells spill over in PB from regenerating marrow? • PB from non-ALL patients on chemotherapy (N: 11) • Range 0 - 0.007 • Mean 0.001 • Median 0 • Statistically similar to normal PB

  27. TdT levels detected in PB from ALL patients at presentation • ALL at diagnosis (N: 38) • Range 0.02 - 165 • Mean 20 • Median 57 • Statistically different from the levels in non-leukemic PB (p: 0.003)

  28. TDT RQ-RT-PCR for MRD detection Sensitivity: 25 leukemic cells in 1,000,000 normal cells (1:40,000) Starting material:0.1ml of blood

  29. TdT RQ-RT-PCR for Diagnosis of CNS leukemia • 20-fold more sensitive than • cytological detection • 50 blasts/ml of CSF (less than 1 blast/μl)