improving diagnosis precision in hematological...
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Improving diagnosis precision in Hematological Malignancies by NGS
Salem Khalil MD, FRCPA, FCAP Consultant Hematopathologist Laboratory Director; Molecular Diagnostic Laboratory/Saudi Diagnostic Laboratory Section Head, Cytogenetic /Molecular Genetics Department of Pathology and Laboratory Medicine (MBC 10) King Faisal Specialist Hospital and Research Centre, Riyadh
MOLECULAR GENETICS
DIAGNOSTIC LABORATORY
Total molecular tests performed
between 1994-2016
>100,000 samples
Department of Pathology and Laboratory Medicine Experience King Faisal Specialist Hospital and Research Center
No Molecular Hematology Test List
1 Sickle Cell Anemia
2
Beta Thalassemia
3 Heriditary Hemochromatosis
4 Factor V Leiden Mutation
5 Prothrombin 20210 Mutation
6 MTHFR Deficiency
7 JAK2 Mutation, Comprehensive
8 BCR-ABL Quantitation
9 BCR-ABL Subtyping
10 PML-RAR-alpha t(15;17), Quantitative PCR
11 Ig Heavy Chain Rearrangement
12 T-Cell Receptor Gamma Rearrangement
13 Chimeric Post-Transplant Graneulocyte and Lymphocyte
14 Chimeric Study Donor
15 Chimeric Study Pre-Transplant Recipient
16 Maternal Cells Engraftment - Granulocyte
17 Maternal Cells Engraftment - Lymphocyte
18 Maternal Cells Engraftment - Mother
19 FMS-Like Tyrosine Kinase 3
20 Nucleophosmin Nucleolar Phosphoprotein B23
22 RUNX1/ETO
23 Isocitrate Dehydrogenase 1, Soluble
24 Isocitrate Dehydrogenase 2, Mitochondrial
25 TEL/RUNX1
26 BCR-ABL Kinase Domain Mutation
27 C/Enhancer Binding Protein Alpha
28 DNA Cytosine -5-Methyltransferose 3 Alpha
29 Wilm’s Tumor
To
tal
Sp
ec
ime
ns
MOLECULAR GENETICS WORK LOAD @ kfsh&rc
98 292 438 518 267
584 915 1048 984
1402 1492 1714 1689
1971 2337
3026 2984
3826
14500
16500
12000
11229
12481
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1994 - 2016
SNP Cytogenomic Array 576 BCR-ABL Kinase Domain Mutation 56 B-raf Gene Mutation 35 BCR-ABL Quantitation 1997 BCR-ABL Subtyping 2 Beta Thalassemia 46 Bone Marrow Cryo Perservation 1208 C\Enhancer Binding Protein Alpha 126 Chimeric Post-Transpland Graneulocyte and Lymphocyte 2376 Chimeric Study Donor 162 Chimeric Study Pre-Transplant Recipient 154 DNA Cytosine-5-Methyltransferose 3 Alpha 104
DNA Extraction and Banking 1268 EGFR 71 FMS-Like Tyrosine Kinase 3 179
Factor V Leiden Mutation 657 Familial Adenomatous Polyposis 16 Hereditary Non-polyposis Colorectal Cancer 0 Hereditary Hemochromatosis 34 Ig Heavy Chain Rearrangement 52 Isocitrate Dehydrogenase 1. Soluble 136 Isocitrate Dehydrogenase 2. Mitochondrial 136 JAK2 Mutation, Comprehensive 711 K-ras Gene Mutation 0 MTHFR Deficiency 550 Maternal Cells Engraftment - Granulocyte 11 Maternal Cells Engraftment - Lymphocyte 11 Maternal Cells Engraftment - Mother 11 Multiple Endocrine Neoplasia Type 1 0 Multiple Endocrine Neoplasia Type 2A and 2B 19 Nucleophosmin Nucleolar Phosphoprotein B23 180 PML-RAR-alpha t(15;17), Quantitative PCR 424 Prothrombin 20210 Mutation 624 RUNX1/ETO 78 Sickle Cell Anemia 64 T-Cell Receptor Gamma Rearrangement 111 TEL/RUNX1 8 Wims Tumor 134 c-Kit Oncogene 154
2016 Molecular Genetics workload 12481 samples
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Molecular Genetics workload 2016 (12481 Samples)
Molecular Testing BCR/ABL P-210 from 2009-2016
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2009 2010 2011 2012 2013 2014 2015 2016
1019
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1861 1812 1949
2233
2045 1997
MagNA Pure LC
The Complete System
Xpert BCR-ABL Monitor
Sanger Sequencer
(1) When to perform mutation analysis? (2) How to perform it? (3) How to translate results into clinical practice?
ABL kinase Domain Mutations
At diagnosis Only in Acceleration Phase (AP)/ Blast Crisis (BC) patients (70-80% of patients harbor mutations)
During first-line Imatinib therapy In case of failure (29% of patients harbor mutations) In case of an increase in BCR-ABL transcript levels leading to MMR loss In any other case of suboptimal response (16% of patients harbor mutations )
During second-line Dasatinib or Nilotinib therapy In case of hematologic or cytogenetic failure
ABL kinase Domain Mutations
When to perform mutation analysis?
ABL kinase Domain Mutations
How to perform mutation analysis?
Direct sequencing is the method recommend for BCR-ABL KD mutation analysis. Direct sequencing may be combined with denaturing-high performance liquid chromatography (D-HPLC) analysis, wherever this technology is available.
Normal Sequence at Codon 315 is ACT
The mutation(T315I) Changes from ACT >ATT
ABL kinase Domain Mutations
How to translate results into clinical practice?
The most appropriate therapeutic options based on the BCR-ABL KD mutation status T315I Ponatinib (Iclusig®) or HSCT V299L, T315A, and F317L/V/I/C Consider Nilotinib rather than Dasatinib Y253H, E255K/V, and F359V/C/I Consider Dasatinib rather than Nilotinib Any other mutation Consider high-dose Imatinib* or Dasatinib or Nilotinib
ABL kinase Domain Mutations > 90
ABL kinase Domain Mutations at KFSH&RC (2012-2016)
Total number of patients :266 patients (2011-2016)
Age range between 10-79 years (median age: 50 years).
Among our patients, 14 % were positive for 15 different mutations
across the ABL1 Kinase domain mutations.
The duration from diagnosis to mutation detection ranged between 3-
144 months with a median duration of 4 years.
Type and frequency of ABL kinase domain mutations
KFSH&RC (2012-2016)
Mutation type No of Patients
M244V 01
L248V 01
Y253H 03
E255K 05
L298 01
V299L 01
T315I 15
F317L 02
Y326H 01
E355G 01
F359I 01
G250E 01
E453K 01
H396K 02
F317I 01
P-loop Sh3
Sh2
contact
A-loop
L248V (1)
M244V (1)
Y253 H (3)
E255K (5)
L298 (1)
V299L (1)
T315I(15) 42%
F317L (2)
Y326H (1)
F359I (1)
E355G (1)
ABL kinase Domain Mutations Department of Pathology and Laboratory Medicine Experience
King Faisal Specialist Hospital and Research Center
(2011-2016)
Gene Mutations
in Acute Myeloid Leukemia Department of Pathology and Laboratory Medicine Experience
King Faisal Specialist Hospital and Research Center
Total number of patients : 192 New AML patients (2012-2016) Median age: 29 years With Average of 31 years
MRN: ________________ BM# : __________________BC#: _________________ DATE: ________________
DIAGNOSIS:
CYTOGENETICS
Karyotype FISH Panel for Hematological Malignancies Neuroblastoma
Diagnostic AML Panel PML/RARA Urgent N-MYC
Follow-up BALL Panel BCR/ABL
TALL Panel C-MYC
MDS Panel
CLL Panel
Myeloma Panel
MOLECULAR GENETICS
PML/RARA [t(15;17)] - PCR Urgent MPN Panel
AML Panel - Mutations BCR/ABL [t(9;22)] - PCR
c-KIT mutation JAK2 Mutation
FLT3 mutation MPL - Mutation
NPM1 mutation CALR Mutation
CEBPA mutation
1DH - 1 mutation TKI - Resistance Mutation
1DH - 2 mutation BCR/ABL Kinase Domain Mutation
WT1 mutation
DNMT3A mutation
Others (please specify) :_________________________________
PATHOLOGIST :______________________________ DATE:______________________
KING FAISAL SPECIALIST HOSPITAL AND RESEARCH CENTREDepartment of Pathology and Laboratory Medicine
Cytogenetics / Molecular Genetics Section
BONE MARROW
AML/FISH Panel
RUNX1/RUNX1T1 Translocation (8;21)
PML/RARA Translocation (15;17)
CBFB (16q22) Rearrangement
MLL (11q23) Rearrangement
RPN1/MECOM Translocation (3;3) or inv(3)
MDS/ FISH Panel
(5q31) EGR1 for -5/5q-
(7q31) D7S522 for -7/7q-
7cen and 8cen
20q12 (D20S108) Deletion
RPN1/MECOM Translocation (3;3) or inv(3)
Pediatric B-cell ALL/FISH Panel
BCR/ABL1 t(9;22) Translocation
MLL (11q23) Rearrangement
4cen, 10cen and 17cen
ETV6/RUNX1(TEL/AML1) for t(12;21) DNA Probe
Adult B-cell ALL/FISH Panel
BCR/ABL1 t(9;22) Translocation
MLL (11q23) Rearrangement
PBX1/TCF3 Translocation (1;19)
CLL/FISH Panel
MDM2/Cen12 for Trisomy 12
13q14 (D13S319)/13q34 Deletion
ATM (11q22.3) Deletion
p53 (17p13.1) Deletion
CCND1/IGH Translocation (11;14)
Multiple Myeloma/FISH Panel
MDM2 (12q15) DNA
13q14 (D13S319)/13q34 Deletion
p53 (17p13.1) Deletion
CCND1/IGH Translocation (11;14)
FGFR3/IGH Translocation (4;14)
MuSiC analysis
Frequency of gene mutations in acute myeloid
leukemia
A total of 192 Cases (2012-2016)
*Data obtained from The Cancer Genome Atlas Research Network, The New
England Journal of Medicine, 30 MAY 2013
Gene International* KFSH & RC†
FLT3 20-30% 09-23%
NPM1 25-35% 12-21%
CEBPA 10-19% 04-05%
1DH-1 06-09% 05-12%
1DH-2 06-12% 07-18%
c-Kit 02-05% 02-03%
WT1 10-13% 08-16%
DNMT3A 18-22% 04-13%
Frequency of gene mutations in acute myeloid Leukemia
A total of 192 Cases (2010-2016)
0%
5%
10%
15%
20%
25%
Significantly mutated genes in AML NPM1 Gene Frequency 25–35% of patients
AML with an NPM1 mutation is a clinicopathological entity. Most frequent in cytogenetically normal AML (45–60% of cases). frequently associated with other mutations (e.g., FLT3-ITD and mutations in DNMT3A, IDH1, IDH2, and TET2) In younger patients, cytogenetically normal AML with mutated NPM1
without FLT3-ITD is associated with a favorable outcome. Older patients (>60 years) with NPM1-mutated AML benefit from
conventional intensive chemotherapy Genetic marker for assessment of minimal residual disease
Significantly mutated genes in AML FLT3-ITD Gene Frequency 20% of patients
Most frequent in cytogenetically normal AML (28–34% of cases)
Associated with unfavorable Tyrosine kinase inhibitors with activity
against FLT3 are in clinical development. (First Generation vs Second Generation)
Significantly mutated genes in AML DNMT3A Gene Frequency 18-22% of patients
Early event in leukemogenesis. Incidence increases with older age. Most frequent in cytogenetically normal AML (30–37% of
cases). When associated with NPM1 and FLT3-ITD mutations a
moderate adverse effect on outcome; possibly limited to the unfavorable ELN molecular subgroup of cytogenetically normal AML.
Associated with clonal hematopoiesis in healthy elderly persons.
Significantly mutated genes in AML
KIT mutation Gene Frequency < 5% of patients
Mostly detected in core-binding factor AML (25–30% of cases)
Confers unfavorable prognosis in AML with t(8;21). The unfavorable effect in AML with inv(16)/t(16;16)
less firmly established. Tyrosine kinase inhibitors with activity against KIT are
in clinical development.
FLT-3 ITD
NPM 1
Molecular Features of MPNs
60%
0%
20%
5% 15%
ET
JAK2V617F
JAK2exon 12
CALR
MPL
Triple negative
59%
0%
24%
7% 10%
PMF
JAK2V617F
JAK2exon 12
CALR
MPL
Triple negative
95%
5%
PV
JAK2V617F
JAK2exon 12
CALR
MPL
FLT-3 ITD
NPM 1
Gene mutations in AML at KFSH&RC 2012-214
2010 to 2013 at KFSH&RC
1811 samples
2014 to 2016 at KFSH&RC
1430 samples
Screened for mutations in exons 12–15 of JAK2 by
Sanger sequencing
JAK-2 Mutation, Year 2010-2016
0
500
1000
1500
2000
2500
3000
3500
Total NEG POS Failed
3241 84%
12%
2855
386
4%
79
• Of the 271 patients with mutations in JAK2: – 262 (96.7%) were positive for the JAK2 p.V617F
mutation
– other JAK2 mutations were identified in the remaining nine (3.3%) patients
JAK/STAT Pathway
In agreement with previous reports, JAK2 p.V617F was the most prevalent mutation detected among our patients (96.7%).
(Kralovics R et al. 2005; James C et al. 2005; Baxter EJ et al. 2005)
The non-p.V617F JAK2 mutations identified were in exons 12 and 13; these corresponded with recently reported mutations, except for the p.I540_N542delinsM mutation
CALR Mutation
• 50-80% of JAK2 or MPL unmutated ET or PMF.
• CALR gene is located in exon 9 of chromosome
19p13.2.
• Encodes Ca+2 binding chaperone (calreticulin)
associated with endoplasmic reticulum.
• Frame shift mutation with insertion or deletion.
• 2 types of CALR mutation(>80% of cases):
– Type I mutation:52 bp deletion.
– Type II mutation:5 bp TTGCT insertion.
Prognostic Significance of MPNs’ Molecular Features
• JAK2V617F, JAK-2 exon 12 mutation and MPLW515 K/L are not
associated with survival or leukemic transformation in PV or ET.
• In ET:
• JAK2V617F associated with increase incidence of thrombosis
and lower risk of post ET-MF.
• CALR mutation associated decrease incidence of thrombosis.
• In PMF: • CALR mutation associated with favorable survival compared to
JAK2 mutation (8.2 vs 4.3 years).
• Triple negative(JAK2-,CALR-,MPL-) associated with unfavorable
survival.
Tefferi A. et al. Blood 2014:124(16):2507-2513
Vannuchi et al Leukemia 2013;27(9):1861-1869
Tefferi A. et al. Leukemia 2014:1472-1477
Triple negative vs. CALR vs. JAK-2 vs. MPL Mutation in PMF
Tefferi et al. Blood 2014;124:2507-2513
• Triple neg (median survival 2.3 yrs.) vs. CALR +PMF(15.9yr) HR 0.2( 95% CI 0.1-0.3). • Triple neg (2.3yr) vs. JAK2 +(5.9 yrs.) PMF HR 0.5 ( 95% CI 0.4-0.7). • CALR +(15.9yr) vs. JAK2+( 5.9yr) PMF HR 2.5 (95% CI 1.7-3.7).
A total of 49 patients have been additionally investigated for CALR and
MPL ; out of these patients only 6 patients ( 12 % ) are positive for
CALR and 2 patients ( 4 % ) are positive for MPL mutation .
The grate part of these patients 41 patients ( 84 % ) are negative for JAK2
, CALR, and MPL mutations " Triple negative ".
Triple-Negative Myeloproliferative Neoplasms
The development of massively parallel sequencing (termed next generation sequencing) revolutionized our ability to analyze cancer genomes.
Massively parallel sequencing results in the generation of millions of short (50-100 nucleotides) DNA sequences simultaneously. Consequently, the majority of sequence variants identified in a cancer genome are inherited polymorphisms and not acquired mutations. Therefore, a comparison of a tumor genome with its paired normal genome is required to efficiently identify acquired (somatic) sequence variants. Currently, there are several different ways in which next-generation sequencing is being applied to study cancer genomes.
Next Generation Sequencing
Construct Library
8 samples ~7 hours
Run Sequence on Ion s5
3-6 hrs
Prepare Template
14-15 hrs
Cloud Alignment and analysis
Up to 6 hrs( connection)
Next Generation Sequencing
ABL1 CYLD FGFR3 KIT PDGFRB TINF2
ASXL1 DID01 FLT3 KRAS PHF6 TP53
BCR DKC1 G6P3 MLL PML TRRAP
BRAF DNMT3A GATA1 MPL RARA U2AF1
C16O4F57 ETV6 GATA2 MYC RUNX1 WAS
CALR EZH2 GATA3 MYD88 SETBP1 WT1
CARD11 FANCA HBB MYH11 SF3B1 ZNF384
CBFB FANCC HRAS NHP2 SH2B3 ZNF521
CBL FNAND2 IDH1 NOP10 SRSF2 ZRSR2
CDKN2A FANC3 IDH2 NOTCH1 TAL1
CEBPA FANCS IKZF1 NPM1 TCAB1
CSFR4 FBXW7 JAK2 NRAS TERC
CSF3R FGFR1 JAK3 PDGFB TERT
CUX1 FGFR2 KDR PDGFRA TET2
King Faisal Specialist Hospital and Research Center
Department of Pathology and Laboratory Medicine
Molecular Genetics Laboratory
NGS Hematology Profile
Classification of somatic variants in tumors:
Class A – This variant is established as clinically
actionable (druggable/predictive/prognostic and/or with
diagnostic/classification implications) in the disease
primary site & histology in which it has been identified.
Class B – This variant is established as actionable in a
different disease site and/or histology; however, in this
site/histology, actionability (or non-actionability) has not
been established
Class C – Variants of this gene in this primary site/histology are established as actionable; however, this specific sequence variant is not one of the recurrently reported variants, (nor is it an established benign single-nucleotide polymorphism) in this gene.
Classification of somatic variants in tumors:
Class D – Variants of this gene in a different primary
site and/or histology are established as actionable;
however, in this site/histology, actionability (or non-
actionability) has not been established, and this specific
sequence variant is NOT one of the recurrently reported
variants (nor is it an established benign SNP) in this
gene.
Class E – Variants in this class are of unknown
significance. No actionability has been established for
any variant in this gene in any disease site/histology.
Classification of the variants regarding functional relevance for protein function (based on international recommendations):
Class 1 – pathogenic variant Class 2 – likely pathogenic variant Class 3 – Variant of Unknown Significance (VUS) Class 4 – likely benign Class 5 – benign
TruSight® Myeloid Sequencing Panel 54 genes (tumor suppressor genes and oncogenic hotspots) in one assay
The Myeloid Tumor Panel (23 genes) for ; MDS and AML The entire coding region of the; ASXL1, CEBPA, DNMT3A, ETV6, EZH2, FLT3, IDH1, IDH2, KIT, KRAS, NPM1, NRAS, PTPN11, RAD21, RUNX1, SF3B1, SMC1A, SMC3, STAG2, TET2, TP53, U2AF1 and WT1.
MOLECULAR DIAGNOSTIC LABORATORY (MDL) 7th. Floor, Burj Al‐Mohammadiyah Building King Faisal Specialist Hospital International Holding Co. King Fahad Road, Al‐Olaya District, RIYADH, Saudi Arabia Telephone: (+966) 1 205 5159 / (+966) 1 205 5162 Facsimile: (+966) 1 205 5171 NGS Acute Myeloid Leukemia Specimen Type Peripheral blood Pathogenic Mutations Detected 1. FLT3: c.2525A>G;p.Tyr842Cys (FLT3-TKD, 48%) No other pathogenic mutations were detected in the other genes tested on the panel. See below for Variants of Unknown Significance and Additional Notes. Please see the section of "Panel Gene List" below for the complete list of genes tested. Interpretation 1. FLT3:c.2525A>G;p.Tyr842Cys (FLT3-TKD) Normal gene/protein function: The FLT3 gene located on chromosome 13q12 encodes the protein FMS-like receptor tyrosine kinase 3 (FLT3), a class III receptor tyrosine kinase (RTK) that plays an important role in hematopoietic stem cell survival, proliferation and differentiation (Kivoi et al, 2002, 12400596). FLT3 activates downstream signaling pathways including RAS, AKT1, ERK, and mTOR (Mizuki et al, 2000, 11090077; Zhang et al, 1999, 10080542; Chen et al, 2010, 21067588). Mutation effect: The p.Tyr842Cys FLT3-tyrosine kinase domain (FLT3-TKD) mutation has been shown to be constitutively activating, and may confer resistance to FLT3 inhibitors when found in conjunction with FLT3-ITD mutations in AML patients (Levis et al, 2013, 24319184;Mead et al, 2007, 17456725). Disease associations: FLT3-TKD point mutations occur in approximately 7% of acute myeloid leukemia (AML) patients, and are less common than FLT3-ITD mutations. Their prognostic impact in AML is less well-defined as compared to FLT3-ITD. They may confer resistance to FLT3 inhibitors (most commonly D835 and F691 variants), when found in conjunction with FLT3-ITD mutations in AML patients (Levis et al, 2013, 24319184; Mead et al, 2007, 17456725). Therapeutic implications: In preclinical studies, some FLT3 inhibitors such as crenolanib, lestaurtinib and midostaurin have shown inhibitory effects on both FLT3-ITD and FLT3-TKD mutations (Levis et al, 2013, 24319184; Grunwald
NGS Acute Myeloid Leukemia Panel