Improving diagnosis precision in Hematological...

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

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

1994

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

2010

2011

2012

2013

2014

2015

2016

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

0

500

1000

1500

2000

2500

Molecular Genetics workload 2016 (12481 Samples)

Molecular Testing BCR/ABL P-210 from 2009-2016

0

500

1000

1500

2000

2500

2009 2010 2011 2012 2013 2014 2015 2016

1019

1300

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


When to perform mutation analysis?


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


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


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


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


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


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

Improving diagnosis precision in Hematological...

Documents

Transcript of Improving diagnosis precision in Hematological...