Mutational Landscape of ALL: Next-Generation Sequencing-based Mutations Scanning Strategy 15 Mar 2019 Seung-Tae Lee Dept. of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Korea WHO 2016 classification B-lymphoblastic leukemia Key genetic subtypes of B-ALL Iacobucci et al. J Clin Oncol 2017 Philadelphia-like ALL • Adverse prognosis • Responsiveness to TKIs • Peak incidence in young adults Iacobucci et al. J Clin Oncol 2017 Common genetic features of Ph-like ALL • Cytokine receptor and tyrosine kinase signaling – CRLF2 mutation (~ 50%) – ABL-class tyrosine kinase gene rearrangement (12%) – JAK2 rearrangement (7%) – EPOR rearrangement (3~10%) – JAK-STAT activating mutation (11%) – Ras signaling (NRAS, KRAS, PTPN11, and NF1; 6%) – kinase alterations (FLT3, NTRK3, BLNK, TYK2, and PTK2B) • B-lymphoid transcription factor genes – IKZF1 deletion Kinase Gene Fusions Philadelphia-like ALL Roberts et al. New Eng J Med. 2014 CRLF2 deregulation • Mutation type – IGH-CRLF2 translocation – P2RY8-CRLF2 fusion (by focal deletion upstream of CRLF2) – CRLF2 point mutations (F232C) • Associated features – Common in Ph-like and Down syndrome–associated ALL – Additional alterations in JAK-STAT (JAK1, JAK2), Ras signaling genes, FLT3, IL7R, SH2B3 and TSLP – Poor prognosis (especially when with IKZF1 deletions) – Therapies targeting JAK-STAT, PI3K/mTOR, and BCL2 signaling show efficacy in preclincal models DUX4- and ERG-deregulated ALL • DUX4/IGH – DUX4 is not expressed in normal B cells – t(4;14)(q35;q32): truncated DUX4 is expressed when translocated to IGH – Truncated DUX4 interact with ERG and produce altered ERG • DUX4/ERG – Truncated ERG is expressed, which inhibits wild-type ERG transcriptional activity, and is transforming. – Favorable outcome, despite concomitant genetic alterations with poor outcomes (e.g. IKZF1, 40% of cases) – t(4;21)(q35;q22): not evident on karyotypic analysis but can be identified by sequencing MEF2D and ZNF384 gene fusions • MEF2D rearrangements – 3~4% of pediatric and 6~7% of adult ALLs – Rearranged to BCL9, HNRNPUL1, SS18, FOXJ2, CSF1R, and DAZAP1 – Older age of onset – Aberrant immunophenotype (CD10-, CD38+) – Poor outcome – Sensitivive to HDAC inhibitors (panobinostat) • ZNF384 rearrangements – Rearranged to EP300, CREBBP, TAF15, SYNRG, EWSR1, TCF3, and ARID1B – Often biphenotypic (B/myeloid) – Up-regulation of JAK/STAT pathway Mutations in relapsed ALL • CREBBP – ~20% of relapsed ALLs – Impair sensitivity to glucocorticoid therapy • NT5C2 – Resistance to purine analogs • MSH6 (mismatch repair) • NR3C1 (glucocorticoid receptor) • SETD2 (H3K36 trimethyltransferase) • KDM6 (lysine-specific demethylase) • MLL2 (epigenetic regulator) • Ras pathway mutations (e.g., KRAS, NRAS, FLT3, PTPN11) Gene mutations in B-ALL Pathway Gene Frequency (%) RAS signaling NRAS 17 KRAS 16 FLT3 7 PTPN11 5 NF1 3 B-cell differentiation PAX5 15 IKZF1 3 JAK/STAT signaling JAK1 2 JAK2 9 TP53/RB1 pathway TP53 4 RB1 1 CDKN2A/CDKN2B 1 Others TBL1XR1 2 ETV6 4 CREBBP 2 Unknown genes 9 Key genetic subtypes of T-ALL Translocation in T-ALL • 14q11 translocation (TCRA, TCRD, TCRB locus) – ~ 50% of T-ALLs – Juxtaposing transcription factor genes, such as TAL1, TAL2, LYL1, OLIG2, LMO1, LMO2, TLX1 (HOX11), TLX3 (HOX11L2), NKX2-1, NKX2-2, NKX2-5, HOXA genes, MYC, and MYB. • STIL-TAL1 fusion – Submicroscopic deletions fuses the promoter of STIL to TAL1 to induce an abnormal expression of TAL1 – ~25% of T-ALL patients • Cryptic rearrangements of ABL1 (NUP214-ABL1, EML1-ABL1, and ETV6-ABL1) – Possible candidate for TKI therapy Gene mutations in T-ALL Pathway Gene Frequency (%) Cell cycle defects CDKN2A/CDKN2B 96 TP53, RB, p27 4 Differentiation impairment TAL1, LMO1/2 39 LYL, LMO2 20 TLX1 7 TLX3 20 HOXA10/11 7 PICALM-MLLT10 5-10 MLL-fusions 4 TAL2 1 Proliferation and survival ABL1-fusions 8 NRAS 5 FLT3 5 LCK 1 ETV6-PBL2, ETV6-JAK2 1 PTEN 1 Self-renewal capacity NOTCH1, FBXW7 56 Pathways deregulated in T-ALL JAK-STAT Ras/PI3K/AKT NOTCH IL7R, JAK1, JAK3, STAT5B NRAS, KRAS, PTEN NOTCH1, FBXW7 Transcription Epigenetic mRNA/ribosome regulator PHF6, SUZ12, EZH2, TET2, CNOT3, RPL5, RPL10 H3F3A, KDM6A LEF1, WT1, BCL11B, ZEB2 Early T-cell Phenytpe (ETP)-ALL Coustan-Smith et al. Lancet 2009; Neumann et al. Blood 2013 Super-enhancer mutation in T-ALL Mansour et al. Science 2014 Goossens et al. Blood 2017 Germ-line predisposition to ALL Stieglitz et al. Therapeutic Advances in Hematology 2013 Clinical Utility of NGS Testing in Acute Leukemias • Diagnosis – Molecular classification (WHO 2016) Recurrent translocation Gene mutation – Precedent disorders Predisposing syndromes • Prognostic risk stratification • Eligibility for targeted therapy • Minimal residual disease (MRD) monitoring NGS panel for ALL (185 genes) B-ALL T-ALL NRAS, KRAS, FLT3, PTPN11, CDKN2A/B, TAL1, LMO1/2, PAX5, IKZF1, JAK2, CREBBP, TLX1, TLX3, NOTCH1, TP53, CDKN2A, etc. etc. ABL1, ABL2, CRLF2, CSF1R, EPOR, ETV6, JAK2, KMT2A, PDGFRB, STIL, TCF3 rearrangements Fusion genes 11 partner genes Validation for ALL panel • 83 patients with ALL – 33 children – 28 adolescent and young adult (AYA) – 22 adults • Classification – 71 B-ALL – 12 T-ALL • Bone marrow samples at initial diagnosis (or in relapse) – Blast > 50% Raw data Bioinformatics pipeline in Align & mapping (BWA) Severance Realignment (GATK) Hospital Normalized GATK Mutect, Varscan IGV Pindel Delly ExomeDepth Custom depth Insertion/ Transloc Genic CNVs Chromosomal Annovar VEP deletion) ation (crosscheck) CNVs SNVs (crosscheck & filtering) In silico Database, literature search (Alamut, HGMD, COSMIC) Oncogenic, likely oncogenic, VUS Correlation with clinical findings Consensus discussion Confirmation (Sanger sequencing, MLPA, microarray) Final report Copy number analysis by read-depth comparison B-ALL • 1.6 (113/71) mutations per patient • 24 patients (33.8%) did not have oncogenic mutation • 45/113 mutations (39.8%) are copy number variations T-ALL • 3.9 (47/12) mutations per patient • All patients had oncogenic mutations • 16/47 mutations (34.0%) are copy number variations A case with ETV6/RUNX1 translocation A case with hyperdiploidy Chromosomal CNVs Off-target analysis On-target DNA Probe Off-target DNA Wash-out ALL with hyperdiploidy Chromosomal CNVs identified by NGS #1 aneuploidy: -2,+6,+7,-8,-16 #29 5q+, 7p-, 9p-,13q-, 15q-, 21?, X? #2 interstitial deletion: 5,6,9,10,19 #30 -9, 17p13.3p11.2-, 1711.2q25.3+,21q11.2122.2+, -X #3 deletion: 1p22.3q21.1, 2q34q36.3, 5q21.1q32, 11q14.2q23.1 #31 5q33.2 interstitial deletion #4 +7p #32 9p21.3p13.2 deletion #5 +17,19,21 #33 11q+, Xq+ #6 deletion: 5q14.3q35.3, 12p13.33p12.2, duplication: 13q21.33q34 #34 hyperdiploidy: +4,+8, +14, +16,+17,+18, +21,+X #7 gain: 4q22q35, 5p15q14, loss: 5q21q35, 21q22, Xp11q28 #35 -7 #8 +12,+20 #36 -7 #9 9p- #37 high hyperdipolidy: 1q+, 4+, 6+, 9+,10+,14+, 17+, 18+, 21+ #10 13q+. 17q- #38 high hyperdipoidy: 3q+, 6+, 10+, 13p-, 14+17+, 21+, X+ #11 +14 #39 17q21q23 duplication #12 +5,+6,+8,+14,+17,+18,+21,+22 #40 6q-, Xq+ #13 12p+.12p- #41 gain: 4,6p,9, 14, 17, 18, 21, X #14 17p-, 22+ #42 ?7p-, +10,+14,+17,+21 #15 9p-,11p- #43 4q22.1q28.3-, 9p24.3p13.3-, 16p12.1q13- #16 +18,+21,+X #44 13q13.3q14.3 deletion #17 +6,+14,+21,+X #45 1q+,16q- #18 8q+,11q- #46 -7,-15,-21 #19 +8, 9p-, 20q-, 21+ #47 +6,+10,+14,+21,+X #20 +21(c?) #48 +4,+6,8q+,+14,+17,+18,+21,+X #21 1q+, 13q-, 17q+,21+ #49 7p-, 9q+, 12q interstitial deletion #22 High hyperdipoidy: +2,+4,+6,+10,+14,+21 #50 8p23p22-, 9p24q22- #23 9p-,12p- #51 gain: 4,6,8, 10, 11,14,18, 21,X #24 aneuploidy #52 loss: 12p13.3p13.1, 13q13q34, gain:17p13.3p13.2 #25 high hyperdiploidy: +4,+6,+8,+10,+17,+18,+21 #53 6q15q23.2- #26 1,13,16,21,22 #54 +8, 9p21p13.2-,+21 #27 8,14,17,21 #55 +21 +X #28 1q+, 4p-,8p-,17p-,17p+, 21++ #56 interstitial deletion: 2 6 Algorithms to detect translocations Probes directed to intronic breakpoints Case Age Type RT-PCR / RNA-Seq DNA algorithm (Delly) Detection of 6 51 B-ALL BCR-ABL1 Detected translocation by 16 15 B-ALL BCR-ABL1 Detected 17 25 B-ALL BCR-ABL1 Not detected DNA sequencing 26 52 B-ALL BCR-ABL1 Not detected 31 16 B-ALL BCR-ABL1 Detected 32 40 B-ALL BCR-ABL1 Detected 35 33 B-ALL BCR-ABL1 Detected • 17/27 (62.9%) 54 73 B-ALL BCR-ABL1 Detected transclocaitons could be 61 64 B-ALL BCR-ABL1 Not detected detected by DNA 62 76 B-ALL BCR-ABL1 Not detected 66 22 B-ALL BCR-ABL1 Not detected sequencing 68 46 B-ALL BCR-ABL1 Detected 69 73 B-ALL BCR-ABL1 Detected 70 72 B-ALL BCR-ABL1 Detected 101 58 B-ALL BCR-ABL1 Detected 5 10 B-ALL ETV6/RUNX1 Detected 3 4 B-ALL ETV6-RUNX1 Detected 13 8 B-ALL ETV6-RUNX1 Detected 25 3 B-ALL ETV6-RUNX1 Detected 28 3 B-ALL ETV6-RUNX1 Not detected 45 3 B-ALL ETV6-RUNX1 Not detected 46 5 B-ALL ETV6-RUNX1 Not detected 78 3 B-ALL ETV6-RUNX1 Detected 80 2 B-ALL ETV6-RUNX1 Detected 7 0 B-ALL KMT2A-AFF1 Detected 85 17 B-ALL P2Y8R-CRLF2 Not detected 84 5 B-ALL PAX5-CBFA2T3, P2RY8R-CRLF2 Not detected Difficulties in targeting introns • Breakpoints occur in introns which are difficult to capture due to repeat regions and variable GC content. Abel et al. J Mol Diagn. 2014 Detection of RNA fusion by NGS Detection of RNA fusion by NGS (Illumina TruSight, ArcherDx Fuxion Plex) ID HemaVision Chromosome Illumina TruSight RNA, ArcherDx Fusion Plex 1 b3a2, Major BCR-ABL1 46,XY,t(9;22)(q34;q11.2)[20] BCR-ABL1 rsa(22;9)(q11.23;q34.12) ABL1-BCR rsa(9;22)(q34.12;q11.23) 2 b3a2, Major BCR-ABL1 46,XY,t(9;22)(q34;q11.2)[20] BCR-ABL1 rsa(22;9)(q11.23;q34.12) ABL1-BCR rsa(9;22)(q34.12;q11.23) 3 b3a2, Major BCR-ABL1 46,XY,t(9;22)(q34;q11.2)[21] BCR-ABL1 rsa(22;9)(q11.23;q34.12) BCR-ABL1