Genomic Profiling in Acute Leukaemias

By:Dr Zubaidah Zakaria

Head Cancer Research Centre

Institute for Medical Research

Outline

• Haemato-oncology tests offered in IMR

• Genomic updates in AML/ALL

• Future

FROM THE MICROSCOPE TO THE NGS

1960 -conventional cytogenetictechniques1) Karyotyping

1980 -molecular cytogenetictechniques1) FISH2) SKY

2007 -microarray cytogenetic techniques1) Gene expression

array2) CGH array3) SNP array4) Cytochip focus

haematology array

Milestones of how the diagnostic tools has evolve in the laboratory

2010 -

Next generation

sequencing

1) Whole

genome

sequencing

2) Whole exome

sequencing

3) Targeted

sequencing

1900 -

morphology

Haemato-oncology diagnostic services in IMR

• Cytogenetic

– Bone Marrow Cytogenetics

– Fluorescence In Situ Hybridisation (FISH)

– Chromosomal breakage study

• Molecular

– Leukaemia Translocation study

– Acute Myeloid Leukaemia Mutation study

– Chronic Myeloid Leukaemia Mutation study

– Chimerism analysis

Cytogenetic and Molecular Haemato-oncology

• Diagnosis

• Prognosis

• Monitoring of treatment

Cytogenetic services in Malaysia

IMR

HKL

Hosp Ampang

UKMSJMC

Prince court

PMC

IPPT

HPP

HUSM

Number of cases: 2014 – 2018

Year

No

. of

case

s

0

500

1000

1500

2000

2500

2014 2015 2016 2017 2018

2015

1780

1913

1760 1730

692561

398 386 369

New cases

Follow-up cases

Type of cases: 2014 – 2018

0

100

200

300

400

500

600

2014 2015 2016 2017 2018

ALL

AML

CML

CLL

MDS

MPN

MM

Lymphoma

AA

Others

No

. of

case

s

Year

Clinical Diagnosis Total No. of Cases* Normal Abnormal

ALL 1193 1010 (85%) 183 (15%)

AML 1328 1121 (84%) 207 (16%)

CML 784 361 (46%) 423 (54%)

CLL 61 57 (93%) 4 (7%)

MDS, MPN, MDS/MPN 1032 928 (90%) 104 (10%)

MM 425 410 (97%) 15 (3%)

Lymphoma 766 730 (95%) 36 (5%)

Others 1468 1317 (90%) 151 (10%)*excluding no spread/inconclusive findings

Chromosomal analysis findings: 2013 – 2017

Lab Activities

Cytogenetic requests from hospitals The bone marrow (BM) samples

Processing of BM samples Chromosome spreading

Lab Activities

Chromosome staining Slides ready for analysis

Loading slides into automated scanner Chromosome analysis & karyotyping

Automated Cytogenetics Platform CytoVision® - GSL120

❖ Image Capturing❖ FISH Scoring

Capacity: 120 slides

ALL – Philadelphia (Ph’) Chromosome

46,XX,t(9;22)(q34;q11.2)

BCR/ ABL1 Dual Fusion Probe

BCR – Chr 9

ABL1 – Chr 22

Fluorescence In Situ Hybridization (FISH) - Interphase

Two signals for

each color in

normal cells

Two red-green

fused signals -

fusion of the ABL

and BCR genes

External Quality Assessment (EQA)

• Australasian Society of Diagnostic Genomics (ASDG)

• Participated since 2007

• Module: Haematological Malignancy

• 3 Parts

– Part 1 – Case Audit

– Part 2 – Statistical Data Collection

– Part 3 – Image Analysis

Molecular Haemato-oncology

• Leukaemia Translocation study

• Acute Myeloid Leukaemia Mutation study

• Chronic Myeloid Leukaemia Mutation study

• Chimerism

Algorithm for leukaemia molecular studies

Leukaemia Translocation Study

• Leukaemia translocation study (Hemavision 28)

– HemaVision kit HV01-28N

– Identifies 28 chromosomal translocations and more than 145 breakpoints associated leukaemia

• Identifies chromosomes, genes, exons at the breakpoint in fusion genes

– Multiplex nested RT-PCR

• Pilot study on childhood leukaemia translocations (HKL): 2008-2011

• Offered for the whole country (paeds and adult) : June 2012 till present

List of 28 leukaemia translocations

Leukaemia Translocations: 2008- 2018

del1 (p32) (STIL-TAL1)

1%

t(1;19) (q23;p13) (TCF3-PBX1)

1%

t(6;11) (q27;q23) (MLL MLLT4)

1% t(8;21) (q22;q22) (RUNX1 RUNX1T1)

4%

t(9;11) (p22;q23) (MLL-MLLT3)

1%

t(9;22) (q34;q11) (BCR-ABL1)

15%

t(12;21) (p13;q22) (ETV6-RUNX1)3%

t(15;17) (q24;q21) (PML-RARA)4%

inv(16) (p13;q22) (CBFB-MYH11) 1%

No translocation67%

n = 5115

Types of Detectable Leukaemia Translocations: 2008- 2018

del1 (p32) (STIL-TAL1)2%

t(1;19) (q23;p13) (TCF3-PBX1)

4%

t(4;11) (q21;q23) (MLL-AFF1)

2%

t(6;9) (p23;q34) (DEK-NUP214)

1%

t(6;11) (q27;q23) (MLL-MLLT4)

2%

t(8;21) (q22;q22) (RUNX1-RUNX1T1)

11%

t(9;11) (p22;q23) (MLL-MLLT3)

2%

t(9;12) (q34;p13) (ETV6-ABL1)

1%

t(9;22) (q34;q11) (BCR-ABL1)

45%t(10;11) (p12;q23) (MLL-MLLT10)

1%

t(11;19) (q23;p13.1) (MLL-ELL)

1%

t(11;19) (q23;p13.3) (MLL-MLLT1)

1%

t(12;21) (p13;q22) (ETV6-RUNX1)

9%

t(15;17) (q24;q21) (PML-RARA)

12%

inv(16) (p13;q22) (CBFB-MYH11)

4%

t(16;21) (p11;q22) (FUS-ERG)

1%

n = 4409

Chromosomal translocations in childhood leukaemia: 2008 - 2011

n=229

• Management of patients with AML relies on genetic tests

that can establish:

– Diagnosis

– Prognosis/ prediction of response to therapy

• Molecular mutations have been analyzed to subdivide

AML into prognostic subsets.

• Offered since October 2013

Acute Myeloid Leukaemia Mutation Studies

– FLT-3

• Identifies both FLT-3 ITD

and FLT-3 D835 mutations

– NPM1

– c-KIT

– CEBPA : PCR and sequencing

• Offered in October 2013

PCR and restrictionenzyme digestion

Real-time PCR

Acute Myeloid Leukaemia Mutation Studies

Algorithm for AML mutation studies

(3.33%)

n = 2667

AML mutation studies : 2014-2018

- Development of in house PCR assays- A total of 199 samples from AML patients were

included in the study. - Mutation analyses were performed using PCR

and sequencing.

Results: - 68 patients were positive for the mutations.- FLT3-ITD mutations were detected in 32

patients (16.1%), followed by FLT3-D835 in 5 (2.5%) and NPM1 in 54 (27.1%).

- Double mutations of NPM1 and FLT3-ITD were detected in 23 cases (11.6%).

- Assays validation were performed using Sanger sequencing

Chronic Myeloid Leukaemia Mutation Study

• Identifies CML patients who develop (secondary) resistance to imatinib• 30% to 50% will have one or more BCR-ABL kinase domain (KD) mutations

• Test offered in IMR: BCR-ABL T315I mutation• Real time PCR

• These patients will be planned for transplant• 3rd generation TKI: ponatinib

• Next plan: Identification of full mutation spectrum of BCR-ABL kinase gene mutations using Sanger sequencing (gold standard)

Map of all the amino acid substitutions in the Bcr-Abl KD reported to be resistant to imatinib

BCR-ABL T315I mutation: 2014 - 2018

n= 404

• A total of 285 patients diagnosed with CML with suspected imatinib resistance were included in the study

• Mutation detection: Qualitative real time PCR

Result:• 15/285 (5.26%) were positive for

T315i mutations• 4 patients in chronic phase• 6 patients in accelerated phase• 5 patients in blast crisis

• Mutation testing is recommended for appropriate treatment strategies to prevent disease progression

• BCR-ABL KD mutations were observed in 23/86 patients (27.6%)

• Fifteen different types of mutations have been identified

• We also discovered that two patients have silent mutation at codon 389 and 401

• Amongst all mutations identified, Y253H is the most common mutation

• Spectrum of mutation varies between patients. – 18/23 patients were found to have single mutation

– 5/23 patients have multiple mutations

• Interestingly, we discovered 3 novel mutations: M290R, K285I and K357T

BCR-ABL KD mutation spectrum in CML patients with IM-resistance – identification of 3 novel mutations: Unpublished data

cDNA position with Nucleotide Change Amino Acid Substitution (Gen

Bank no. X16416 (ABL)

No. of

Patients with

Mutation,

n=23

ABL Kinase domain

Single Mutation

757 T>C Y253H 6 P loop

763 G>A E255K 1 P loop

799 A>G T267A 1 n/a

931 T>A F311I 4 IM binding site

944 C>T T315I 2 IM binding site

951 C>A F317L 1 IM binding site

1075 T>G F359V 1 C loop

1075 T>A F359I 1 C loop

1203 C>T none 1 none

Multiple Mutation

757 T>C & 869 T>G Y253H / M290R* 1 P loop/ n/a

757 T>C & 1167 A>G Y253H / None 1 P loop/None

854 A>T & 1375 G>A K285I* / E459K 1 n/a / other mutation

1070 A>C & 1076 T>G K357T*/ F359C 1 C loop

757T>C, 859 G>A & 1195 G>A Y253H / A287T* / A399T 1 P loop/ n/a / A loop

BCR-ABL KD mutation spectrum in CML patients with IM-resistance

*novel mutation

Results

Chimerism study

• A measure of the number of donor and recipient cells in the host following

haematopoietic stem cell transplantation (HSCT)

• Prediction of:Disease relapse

Graft rejection

Graft-versus-host disease

• Test offered in IMR: Quantitave Real time (qPCR)

• Pilot study - HKL in 2010

• Test offered for:

– HKL in 2011 – 2013 (before taken over by HKL Haematology lab)

– Hosp Ampang in 2014

– Hospital Pulau Pinang in 2016

Chimerism study

CHIMERISM STUDIES: 2017 - 2018

Method used: Quantitative real-time PCR

◦ KMRdx (multiplex PCR)

0

1000

2000

3000

4000

5000

6000

7000

Genotyping Quantitation of Chimerism

Workload: Number of Tests performed for chimerism studies

2017 2018

2017 2018

Genotyping 4290 5928

Quantitation of Chimerism

272 194

• Chimerism of blood samples from 57 paediatric patients were analyzed by STR-PCR and qRT-PCR on different days after allogeneic hematopoietics stem cell transplantation.

Results:• Good correlation between the two

methods with correlation coefficient (r)=0.854 (p<0.001).

• High correlation between STR-PCR and qRT-PCR methods helps to validate the use of qRT-PCR for chimerism analysis.

• qRT-PCR has better sensitivity with detection limit of less than 0.1% depending on the amount of DNA used, compared to STR-PCR detection limit of 1-5% only.

• Thus, qRT-PCR is a valid, more sensitive, simple and rapid compared to STR-PCR method in chimerism monitoring.

Genomic updates in acute leukaemias

WHO classification

• A closer integration of morphology and genetics• Generate new diagnostic approaches• Improve prognostic/predictive models• Therapeutics

WHO 2001 WHO 2016

PRECISION MEDICINE

Genomics in Acute Myeloid Leukaemia

• Highly heterogenous disease

• Over the past 10-15 years – virtual explosion in identification of genes and mutations

• Enhanced understanding of the biology and identification of potential targets for therapy

• Biologically and prognostically different subtypes

• Distinguished by cytogenetic and molecular genetic analysis

WHO Classification 2016

AML and related precursor neoplasms

AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1 AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11 Acute promyelocytic leukemia with PML-RARA

AML with t(9;11)(p21.3;q23.3); MLLT3-KMT2AAML with t(6;9)(p23;q34.1); DEK-NUP214 AML with inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2); GATA2,MECOM(EVI1)AML (megakaryoblastic) with t(1;22)(p13.3;q13.3); RBM15-MKL1

AML with mutated NPM1AML with biallelic mutations of CEBPA

Provisional entity: AML with BCR-ABL1

AML with mutated RUNX1

AML with recurrent genetic abnormalities

Considered acute leukaemiasregardless of blast count

Full entities, favourable prognosis

de novo AML , no evidence of CMLmight benefit from TKI therapies

associated with inferior outcome and worse overall survival

Genomic landscape of AML

The Cancer Genome Atlas (TCGA) Research Network. N Engl J Med., 2013;368(22): 2059-74

Analysis of 200 de novo AML patients according to whole genome or whole-exome sequencing: • identified an excess of

200 recurrently mutated genes

• of which 23 were significantly mutated

Molecular classes of AML and concurrent gene mutations

Risk stratification by genetics – European LeukaemiaNet 2017

Genomics in Acute Lymphoblastic Leukaemia

• This group is characterized by recurrent genetic abnormalities, including balanced translocations and abnormalities involving chromosome number

• Prognostic implications

B lymphoblastic leukaemia/lymphoma2 new provisional entities:

• B lymphoblastic leukaemia/lymphoma, BCR-ABL1-like– Translocations involving other tyrosine

kinase inhibitors

• CRLF2, EPOR, PDGFRB etc

– Associated with adverse prognosis

– Respond to some TKI therapies

• B lymphoblastic leukaemia/lymphoma, with iAMP21– Associated with adverse prognosis

B lymphoblastic leukaemia/lymphoma, BCR-ABL1-like

• More recently, a variant with a similar gene expression profile to Ph-positive ALL but without the BCR-ABL1 rearrangement has been identified.

• In more than 80% of cases of this Ph-like ALL → the variant possesses deletions in key transcription factors involved in B-cell development:

– IKAROS family zinc finger 1 (IKZF1)

– transcription factor 3 (E2A)

– early B-cell factor 1 (EBF1)

– paired box 5(PAX5).

• Similarly, kinase-activating mutations are seen in 90% of the Ph-like ALL.

• The most common of these include rearrangements involving:– ABL1, JAK2, PDGFRB, CRLF2 and EPOR, activating mutations of IL7R

and FLT3 and deletion of SH2B3, which encodes the JAK2-negative regulator LNK.

• This has significant therapeutic implications:– tends to carry a worse prognosis

– may respond to kinase inhibitors.

B lymphoblastic leukaemia/lymphoma, with iAMP21

• Characterized by amplification of a portion of chromosome 21

– characteristically detected by FISH with a probe for the RUNX1 gene that reveals 5 or more copies of the gene (or 3 or more extra copies on a single abnormal chromosome 21 in metaphase FISH)

• It occurs in about 2% of children with ALL, especially older children with low WBC counts.

• Uncommon in adults.

• This new entity is associated with an adverse prognosis which can, to some extent, be overcome with more aggressive therapy

Ongoing research project:Precursor B-ALL Genetic Characterization

- unpublished data

HIGH RESOLUTION SINGLE NUCLEOTIDE POLYMORPHISM ANALYSIS OF GENOMIC ABERRATIONS IN CHILDHOOD B ACUTE

LYMPHOBLASTIC LEUKAEMIA

Zubaidah Zakaria1; Nor Soleha Mohd Dali1; Nursaedah Abdullah Aziz1; Lailatul Hadziyah Mohd Pauzy1; Julia Abdullah1; Nor Rizan Kamaluddin1; Ezalia Esa1; Jeyanthy Eswaran2; Yuslina Mat Yusoff1

1HAEMATOLOGY UNIT, CANCER RESEARCH CENTRE, INSTITUTE FOR MEDICAL RESEARCH, JALAN PAHANG, 50588 KUALALUMPUR, MALAYSIA;

2NEWCASTLE UNIVERSITY UK/MALAYSIA, NORTHERN INSTITUTE FOR CANCER RESEARCH, NEWCASTLE UNIVERSITY,NEWCASTLE UPON TYNE, UNITED KINGDOM

• The strong association of many chromosomal abnormalitiesand prognosis has been utilised in risk stratification fortreatment in large number of protocols worldwide.

• In this study, we systematically characterised the genomicaberrations in 61 childhood BCP-ALL patients among Malaysianpopulations.

High Resolution Single Nucleotide Polymorphism Analysis of B-ALL cohort using Copy Number Array SNP6

High Resolution Single Nucleotide Polymorphism Analysis of B-ALL cohort using Copy Number Array SNP6

• Genome wide copy number variations

• B-ALL Subtype classification and Stratification

• Focal deletions and gains

• Complete characterisation – Possible Novel SNPs

• Explore more efficient and cost effective tests

• Test whether known abnormalities are present in Malaysian cohort

• Beginning of a Well Characterised BCP-ALL Cohort in IMR

METHODS

Genomic DNAs extracted from bone marrow

Bone marrow cell suspension

Single Nucleotide

Polymorphism (SNP) Array

using Affymetrix® Genome-Wide

Human SNP Nsp/Sty 6.0

Multiplex Ligation-

Dependent Probe

Amplification (MLPA) using

P327 and P335 kits

Raw data analysis using

Genotyping Console Software

v4.2.0.26 & Chromosome

Analysis Suite v3.3.0.139

Raw data analysis using

Coffalyser software

v140721.1958

Fluorescence In Situ Hybridization

(FISH) using:

➢ CRLF2 breakapart probe,

➢ ETV6/ RUNX1 ES dual colour,

translocation probe,

➢ MLL dual colour, breakapart

rearrangement probe

➢ Centromere probe of chromosome 4,

10, and 17 (XCE 4/ 10/ 17)

Analysis using CytoVision® software

v7.4 build 242

The graph showing total number of CNVs detected by SNP6.0 array. Blue represents Gain; Red represents Loss. Region22q11.22 has the highest number of gains (50/55; 86.2%) (blue arrow), while region 4q13.2 has the highest number oflosses (28/58; 48.3%) (red arrow).

14

35

2

7

11

28

35 35

50

2427

1417

28

18

1012

1 10

10

20

30

40

50

60

1p36.33 2p11.2 3q26.1 4q13.2 8p11.2 14q11.2 14q32.22 15q11.2 22q11.2 Xq21.31 Xp22.33

Graph of Copy Number Variant (CNV) Regions

Chromosome Gains/Loss from SNP6 analysis

Chromosome Gains Chromosome Loss

Number of cases Number of cases

SNP6 Gains and Losses confirmed by MLPA using P335 kit

0 5 10 15 20 25 30

CDKN2A/B

MLL

IKZF1

PAX5

RUNX1

ERG

CRLF2

SHOX

CSF2RA

BTG3

ETV6

3

1

21

21

20

27

20

20

1

18

1

6

14

3

1

1

1

13

Frequency of gain and loss among leukaemia-related genes

Bar graph shows the frequency

of gain and loss among

leukaemia-related gene. Blue

represents Gain, while red

represents Loss.

The most number of gain

occurs at the SHOX gene

(Xp22.33; PAR region) (49%,

n=55) (blue arrow) while the

most number of loss occurs at

the CDKN2A/B gene (9p21.3)

(33%, n=55) (red arrow).

B-ALL Stratification Marker

Gains and Deletions

Study Conclusion

• Determining the genetic landscape of BCP-ALL using SNP6 allows us to establish suitable diagnostic and treatment strategies

• Integrating Clinical Data along with the Genetic Data of BCP-ALL is critical to evaluate treatment outcome

• As a first Step, 2016 BCP-ALL Cohort genetic characterisation is complete and further mutational studies on BCP-ALL are on going as collaborative projects.

Where are we going next?

Next generation sequencing• The application of high-throughput, massively parallel sequencing

technologies • Whole genome sequencing

• Provides a comprehensive overview of the entire 3 × 109 (3 Gbases) human genome at single nucleotide level resolution

• Covers both coding and noncoding regions.

• Whole exome sequencing• Analyzes the protein-coding regions of the genome• Sequencing is restricted to exonic regions of the genome where pathogenic

mutations are more common

• Targeted sequencing• Allows for the selective enrichment of particular genes or regions of

interest• Ideally used in the diagnostic laboratory for the evaluation of malignancies

where the spectrum of mutations is well established and discovery work is not required.

• We identified a total of 16466 somatic mutations

• A total of 86 variants located at the leukaemia-related genes

• 32 variants in the coding region of GL12, SP140, GATA 2, SMAD5, KMT2C, CDH17, CDX2, FLT3, PML, and MOV10L1

Conclusion:

• One or more secondary genetic alterations are required for leukemogenesis

• Detection and identification of secondary genetic alterations are important to identify new therapeutic targets

Poster presentation; College of Pathologist Annual Scientific Meeting 2016

A total of 16 bone marrow and peripheral blood samples of cytogenetically normal AMLpatients

Targeted sequencing using a panel of 20 frequently mutated genes in AML (Agilent ClearSeqAML)

Captured DNA was then sequenced on Illumina MiSeq platform.

Data analysis and bioinformatics were performed using Agilent’s Surecall software.

• Identified 14/20recurrent mutatedgenes in AML.

• All 16 patientsharboured NRAS,SRSF2, SETBPI andASXL1 mutations.

CONCLUSION:➢ Targeted sequencing focuses on a region of interest and restrict analyses on potentially clinically

relevant genes.➢ A practical approach to identify mutational patterns that can further improve diagnostics and

prognostication.

RESULTS:

Facilities

IMR Setia Alam

Nucleic acid extraction

PCR mastermix preparation

PCR Thermal cycler Bioanalyzer

Real-Time PCR system (LightCycler 480)

Real-Time PCR system (ABI 7500)

Gel electrophoresis system and gel documentation

Agilent Microarray Scanner

Illumina MiSeq Next Generation Sequencer

Application on Miseq• Targeted Cancer Panel Validation

– “Lab Developed Test” for Malaysian.

– Myeloid panel (covers AML, MDS, CML, CMML, JMML)

– BRCA panel

– Solid tumor panel (lung, colon, breast, ovarian, melanoma and more)

• Thalassemia Panel

– For early detection of Thala carrier to reduce incident of child birth affected by thalassemia.

– Incorporation of genetic testing in Thalassemia prevention plan helps government to identify the real carrier without guessing

• Cardiovascular inherited condition panel

– early detection and health management plan to delay disease on-set for high risk group

– Carrier will be identified, and seek for consultant of Cardiologist to prepare a heart health monitoring plan

– reduce incidence of sudden cardiac arrest

• Custom Panel of interest

– any other disease of interest?

– wanted more discovery in single testing?

– consolidate molecular testing, reduce workload and improve TAT?

Data output : 300 Mb – 15 Gb

Application on NextSeq500• Large Cancer Panel Validation for Trusight Oncology 500

– Targets on tumor mutational burden and microsatellite stability (523 cancers relevant genes)

– Suitable for myeloid, lung, colon, ovarian, breast, gastric, bladder, melanoma and sarcoma.

– For treatment selection.

– “Lab Developed Test” for Malaysian.

• Cancer Exome Sequencing (All cancer types)

• Cancer Transcriptome Sequencing (All cancer types)

• Rare Disease Diagnosis

– Exome sequencing of infants with undiagnosed condition to identify disease causing mutation for the preparation of treatment plan

– Help patient`s family to know the root cause of the disease and enable family to plan on how to manage a better quality of life for the patient

• Non-invasive Prenatal Testing (NIPT)

– Sequencing of the fetus DNA in maternal blood for early detection of Trisomy 13, 18 and 21.

– Benefit for B40 community pregnant women for maternal health and family planning

Data output : 19.5 Gb – 120 Gb

Conclusion

• Molecular technologies and understanding of genetics have introduced new insights to understanding of acute leukaemias and haemato-oncology

• Advances in molecular genetics will generate new diagnostic approaches, improved prognostic/predictive models and therapeutics/targeted therapy for precision medicine.

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