ADRL Advanced Diagnostics Research Laboratory John DeCoteau, MD FRCP Department of Pathology, Division of Hematopathology University of Saskatchewan Saskatchewan Cancer Agency
ADRL Project Objectives New initiative of the Saskatchewan Cancer Agency (SCA) Mandate is to develop new and emerging molecular and flow cytometry assays for patients with hematological malignancies and solid tumors Tests developed and performed in an accredited environment SOPs, Reporting Templates, QA/validation data Newly developed and validated assays implemented as clinical tests High complexity testing available to support research endeavors Basic science and translational studies Clinical Trials/POR
Capital Equipment Instrument System Real Time PCR Genetic Analyzer (CE) ION Torrent PGM (NGS System) Applications Translocation Detection Quantitative Transcript Monitoring Mutation Detection Mutation Detection Sequencing Confirmation Somatic Hypermutation Engraftment Monitoring (Chimerism) Microsatellite Instability 50-400 Cancer Gene Mutation Panels Somatic Hypermutation (SHM) Gallios 10 Color Flow Cytometer Astrios 10 Color Cell Sorter Leukemia Immunophenotyping, MRD, Immune Monitoring, PNH Engraftment Monitoring (Chimerism)
Next Generation Sequencing Ion Torrent Semiconductor Sequencing >400,000 sequences at a time by NGS vs. 1 48 at a time by CE Amplicon Generation Bead Ligation Clonal Amplification of Template
Next Generation Sequencing Ion Torrent Semiconductor Sequencing
Next Generation Sequencing Ion Torrent Semiconductor Sequencing
Ion Torrent 400 Cancer Gene Panel AML 43 NPM1 (c.861_862ins TGCT); FLT3 ITD
Bar Coding/Multiplexing Reads per chip = 400,000 500,000 Recommended reads per sample = >10,000 400,000 / 20,000 = 20 samples per chip
Batch Testing Myelofocus Panel FLT3 ITD and 835/TKD; NPM1; JAK2 V617F and exon 12; Kit exon 8 and exon 17 (D816X); WT1 exon 7 and 9; NRAS codons 12, 13, 61; KRAS codons 12, 13, 61; ASXL1; IDH1 exon 4; IDH2 exon 4; SETBP1 exon 4; MPL exon 10; CBL exon 8; LBL exon 9; TET2; P53; DNMT3a; PDGFRa; ABL1; RUNX1; CEBPa; MN1; GATA2; PTPN11 Non-Hematopoietic Cancer Panel KRAS, NRAS, HRAS, BRAF, EGFR, P53 Custom/Research Panels
Antigen Receptor Genes Analysis by NGS Harness the power of NGS to create high throughput diagnostic and monitoring assays for lymphoid malignancies Started with an assay that provides important prognostic information but that has shortcomings and is challenging to implement in routine clinical practice Immunoglobulin Heavy Chain Variable Gene (IGHV) Mutation Assay
Conventional IGHV Mutation Assay Patient DNA Amplified IGHV DNA (one patient) Patient A B C Patient A Patient B Patient C re-sequence? sub-clone? Data Analysis (single seq. reads) Reporting /Diagnosi s
IGHV Mutation Status by Conventional Sequencing
Next Generation IGHV Mutation Assay Patient DNA Patients A to Z Amplified IGHV DNA (many patients) OPTIONAL Patient A B C Next- Generation DNA Sequencing High- Throughput Data Analysis (1000s seq. reads) Reporting /Diagnosi s
IGHV Consensus Sequence by NGS
IGHV Mutation Status by NGS Unmutated Mutated
IGHV Mutation Test Comparison IGHV test Time Patients/test Sequences/ patient Reagent cost/patient Conventional 3 days One ~10 $100-200 ADRL NGS 3 days 10-20 > 10,000 $ 10-20 Mutated Gray Zone Unmutated
Clonal Profiling DNA sample IGHV identified (> 5%) IGHV clonal frequency % identity to germline Bone Marrow (MCL) Eyelid Biopsy (MZL+ MCL) IGHV1-2 31.2% 99% IGHV3-7 30.3% 96.8% IGHV1-2 12.8% 99% Each lymphocyte has a genetic fingerprint NGS can be used to sequence all the B-cell receptor genes in a heterogeneous population Frequency data can be used to determine the relative abundance of B-cell clones
CLL Genetic Markers by NGS Gene Sequenced Variant Classification Disease (reference) Patient 1 ATM Gly2925Ala deleterious CLL (COSMIC) BIRC3 Gln547fs deleterious CLL (published) P53 Gly206Ser deleterious CLL, ALL, etc. (COSMIC SF3BP1 Gly742Asp deleterious CLL (COSMIC) NOTCH1 none n/a n/a IgHV Unmutated Patient 2 ATM Gly2023Arg deleterious CLL (COSMIC, published) NOTCH1 Asn151Asp unknown n/a BIRC3 none n/a n/a SF3BP1 none n/a n/a P53 none n/a n/a IgHV Mutated
IGHV Mutation Testing by NGS Simplified workflow with higher patient throughput High statistical confidence 10,000 sequences per patient High sensitivity to detect sequence variations and heterogeneity Flexibility NGS SHM can be performed concurrently with other CLL tests (including p53, ATM, NOTCH1, SP3B1, BIRC3) Strong research potential Deep sequencing more informative and drives hypotheses
10 Color Flow Cytometry Reduced sample requirements Fewer tubes Less time to acquire data Routinely obtain >500,000 cells for MRD analysis from PB or BMAs Rare populations can be unambiguously identified from the background expression of individual markers MRD populations e.g. CLL; AML; ALL Rare normal cell populations e.g. NK T cells; dendritic cells
CLL MRD
CLL MRD CD5 pos CD20 dim
CLL MRD CD5 pos CD20 dim CD43
CLL MRD CD5 pos CD20 dim CD43 CD22
CLL MRD CD5 pos CD20 dim CD43 CD22 CD79b CD81
CLL MRD CD19pos/CD5pos/CD3neg (CLL) > CD19pos/CD5pos/CD3pos 130 CLL cells detected amid 415,555 WBC (0.04%)
CLL MRD
ADRL Requisition CLL WORK UP FLOW CYTOMETRY 45, 3, 5, 19, 20, 22, 23, 200, κ, λ 45, 3, 5, 19, 20, 22, 38, 43, 79b, 81 NGS MUTATION PANEL IgHV Somatic Hypermutation NOTCH1, p53, ATM, BIRC3, SF3B1 Expanded mutation screen
ADRL SUMMARY Develop new and emerging flow cytometry and molecular assays Translate validated assays into the clinical arena through strong linkages with the existing Saskatoon Health Region laboratories Provide high complexity testing to support collaborative research Develop diagnostic reagents in collaboration with the Saskatchewan Therapeutic Antibody Resource (STAR)
Acknowledgements Ron Geyer Karen Mochoruk Landon Pastushok Wayne Hill SCA Scott Livingstone Choc lacure Foundation David Sheridan Julie Stakiw