Can Cytogenetics and FISH survive in the modern genomic era?

Application of Cytogenetic, FISH and Microarray Analysis in Diagnosis of Leukemia and Lymphoma

Yanming Zhang M. D.

Associate Professor, Department of Pathology,

Medical Director, Cytogenetics Laboratory,

Northwestern University Feinberg School of Medicine

Cytogenetics Laboratory at Northwestern Memorial Hospital, Northwestern University

• State-of-the art clinical cytogenetics laboratory with CLIA and CAP certification .

• Opened on October 3, 2011, with an average case load of 2000 hematological

neoplasms and 150 breast, brain and lung cancer samples (PET FISH).

• Staffed with 8 technologists, one resource coordinator, one technical

coordinator, one manager and one medical director.

• Techniques:

Conventional cytogenetic analysis

Fluorescence in situ hybridization (FISH)

Paraffin embedded tissue (PET)-FISH

Genomic SNP microarray

Clinical case for cytogenetic analysis41-year-old woman with a newly diagnosed acute leukemia.

Acute myeloid leukemia with maturation (FAB M2)Myeloblasts: CD34+, CD117+, MPO+, CD13+, CD33+; negative for all lymphoid antigens.

ISCN: 46,XX,t(8;21)(q22;q22)[19]/46,XX[1]

FISH analysis with the AML1/ETO-DF probe

94% of cells show a dual-fusion signal pattern, i.e.

the AML1/ETO fusions

Acute myeloid leukemia with t(8;21)(q22;q22)

45,X,-Y,t(8;20)(q22;p13),del(11)(q21q25)[18/20]

FISH with AML1/ETO –DF

probe:

76% of cells show one fusion, two red and two

green signals

Three way translocation of t(8;21)(q22;q22)

45,X,-Y,t(8;20;21)(q22;p13;q22),del(11)(q21q25)

Procedure of cytogenetic

analysis

• Samples: bone marrow (aspirate or core) (fresh!) peripheral blood

lymph node/spleen/tonsilsolid tumor massCNS, plural fluids, etc

• Culturing: no mitogens addedshort term cultures (24hr, 48hr)

• Chromosomes: Leukemia cells with poor morphology and few short and fuzzy bands, whereas normal cells nice bands.

• Analysis: Heterogeneous populations (normal, abnormal clones).

Precise hematopathological diagnosis is important for targeted detection of recurring chromosome abnormalities in specific subtypes.

Cancer Cytogenetics

Recurring Chromosome Abnormalities in Cancer Cells

• Gains or Duplications

• Losses or Deletions

• Amplifications - Double Minutes (DM) or

Homogeneously Staining Regions (HSR)

• Markers

• Translocations and Inversions

• Acquired Somatic Mutations

• Present in the Malignant Cells

• Clonal

• Nonrandom

t(15;17)(q22;q11.2)APL

t(9;22)(q34;q11.2) ALL/CML

t(14;18)(q32;q21)FL

t(11;14)(q13;q32)MCL

t(8;14)(q24.1;q32)Burkitt Leuk./NHL

Identified by Dr. Janet D. Rowley in 1973 as the first recurring Identified by Dr. Janet D. Rowley in 1973 as the first recurring translocation in acute leukemia. translocation in acute leukemia.

Associated with AML-M2 (~30% of AML-M2 cases, or ~5-10% Associated with AML-M2 (~30% of AML-M2 cases, or ~5-10% of all AML).of all AML).

Characterized by a good response to therapy (98% CR) and a Characterized by a good response to therapy (98% CR) and a prolonged disease-free survival.prolonged disease-free survival.

t(8;21) in AML

Characteristic morphology:Characteristic morphology: myeloid blasts with indented nuclei. myeloid blasts with indented nuclei. basophilic cytoplasm with few basophilic cytoplasm with few

azurophilic granules.azurophilic granules. increased eosinophils in bone increased eosinophils in bone

marrow.marrow. Aberrant expression of CD19, and Aberrant expression of CD19, and

CD56.CD56.

AML1/(RUNX1)• The AML1/(RUNX1) gene at 21q22 codes for core binding factor

(CBF) which forms a heterodimer with CBF that acts as a transcriptional activating factor.

• CBF is a critical regulator in the generation and differentiation of definitive hematopoietic stem cells.

t(8;21)AML1-ETO10% AML

t(3;21)AML1-EVI1

Rare cases of CML and MDS

t(12;21)TEL-AML1

25% pediatric ALL t(16;21)AML1-MTG16

rare cases of AML

inv(16)CBF-MYH11

8% AMLPoint

Mutation10%

MPO, CSF-1R, TCR, IL-3, GM-CSF

---TGTGGT------TGTGGT---

CBF

AML1

Core enhancer sequence

Target genesTarget genes

1. Deregulated expression of a normal protein

2. Production of a fusion protein2. Production of a fusion protein

Consequences of chromosome translocationsConsequences of chromosome translocations

Increased expression of c-MYC

Promoter of IgH Coding regions of c-MYC

Coding regions of AML1 Coding regions of ETO Expression of a fusion

protein AML1-ETOAML1-ETO

t(8;14)

t(8;21)

ETO-AML1

Hematopoietic cell differentiation and chromosome abnormalities in leukemia and lymphoma

hematopoietic stem cell

Mast cell

Erythrocytes

Platelets

Eosinophil

Neutrophil

Monocyte

ALL/NHLt(14q11.2)

t(7q34)

CML t(9;22)

ALLt(12;21)t(1;19)t(8;14)

Hyperdipl.

ALLt(9;22)

t(11q23)

AMLt(8;21) inv(16)t(15;17)

NHLt(8;14), t(14;18)

t(11;14)

AMLt(11q23)

B cell T cell

Myeloid progenitor

Lympho-myeloid Stem cell

Lymphoid Progenitor

Pro BPro B Common BCommon B Pre BPre B B B

ImmunophenotypeImmunophenotype

CD19CD19 ++ ++ ++ + +

CD10CD10 -- ++ + +

CD34CD34 +/-+/- + (most)+ (most) +/-+/-

Ig MIg M -- -- + (cytopl)+ (cytopl) + (surface)+ (surface)

Cytogenetic PatternCytogenetic Pattern

t(4;11)t(4;11) t(12;21)t(12;21) t(12;21)t(12;21) t(8;14)t(8;14)

hyperdipl. (>50) hyperdipl. (>50) t(1;19)t(1;19) t(2;8)t(2;8)

t(9;22) t(9;22) hyperdipl.hyperdipl. t(8;22)t(8;22)

Recurring Chromosome Abnormalities in B-ALLRecurring Chromosome Abnormalities in B-ALL

Insidious (t-MDS)Insidious (t-MDS)

CytogeneticsCytogenetics 11q23, 21q2211q23, 21q22

LatencyLatency 5-7 yrs5-7 yrs 2-3 yrs2-3 yrs

PresentationPresentation acuteacute

PrognosisPrognosis poorpoor poorpoor

Alkylating agents Alkylating agents RadiationRadiation

Topo II inhibitors Topo II inhibitors (VP16, Dox)(VP16, Dox)

Features of therapy-related AMLFeatures of therapy-related AML

-5/del(5q)/-7/del(7q)

MRC/NCRI AML Trials: Overall Survival ages 16-59, 2550 patients, 10 years follow-up

-7/del(7q), n=336

t(15;17), n=607

t(9;11), n=61

t(8;21), n=421

inv(16)/t(16;16), n=284

Inv(3)/t(3;3),n=69

t(3;5), n=25

t(6;9), n=42

-5/del(5q), n=258

AML/MDS, n=343other 11q, n=60

t(9;22), n=44

Years from entry

% a

live

Grimwade et al., Blood, April 12, 2010* Normal karyotypes: 38% OS

• Diagnosis and differential diagnosis:

WHO classification based on specific cytogenetic/molecular genetic findings, such as t(8;21), t(15;17), inv(16), t(9;11) and other 11q23/MLL, inv(3)/t(3;3), t(6;9), t(1;22).

• Treatment protocols:

APL: PML/RARa: ATRA+CT.

CBF [t(8;21) and inv(16)]: HDAC consolidation.

• Monitoring response and engraftment of BMT

cytogenetic complete remission (CR) and MRD

• Prognosis: most critical and independent indicators.

favorable (55-81% cured): t(15;17), inv(16), t(8;21);

intermediate (40%): t(9;11), normal karyotype;

unfavorable (<5%): complex, abnl 5 and 7, inv(3), t(6;9)

Clinical significance of chromosome abnormalities in leukemia and lymphoma

Fluorescence in situ hybridization (FISH)

Three types of FISH probes:

• Centromeric probes: trisomy/monosomy

• Locus specific probes: gain or loss, and translocations.

• Chromosome or arms/bands painting probes: structural abnormalities (SKY, M-FISH).

FISH signal patterns Dual Fusion pattern: highest sensitivity

BCR/ABL-DF AML1/ETO-DF PML-RARa-DF

MYC/IGH-DF BCL1/IGH-DF IGH/BCL2-DF IGH/MALT1-DF

t(9;22)

t(9;22),+Ph

BMTX and Y chromosomes

AMLt(8;21) ETO/AML1t(15;17) PML/RARαInv(16) CBFβ11q23/MLL17q21/RARαinv(3)/t(3;3)

Paraffin-EmbeddedTissue (PET)HER2/CEP17del(1p)/del(19q)PTEN/CEP10EGFR/CEP7ALKEWSR1SS18FOX01

NHLt(8;14) MYC/IGHt(11;14) CCND1/IGHt(11;18) API2/MALT1t(14;18) IGH/BCL2t(14;18) IGH/MALT13q27/BCL68q24/MYC11q13/CCND114q32/IGH18q21/BCL218q21/MALT12p23/ALK

MDS/MPNdel(5q)del(7q)/-7trisomy 8del(13q)/-13del(20q)t(9;22) BCR/ABLdel(4q12)/CHIC2

CLL Panelt(11;14), ATM/11q,CEP12, del(13q),del(17p), 14q32/IGH, MYB/6q23

MM Panelt(11;14), del(13q),del(17p), 14q32/IGH

ALLt(9;22) BCR/ABLt(12;21) TEL/AML1+4/+10/+17del(9p)/p16/CEP911q23/MLL

FISH test menu at NMH Cytogenetics Laboratory

Sample types and preparation for FISH

• Bone Marrow

• Peripheral blood

• Lymph node

• Tumor mass

• CSF

• Plural fluid

• Fresh BM/PB/LN

• Cytospin slides

• BM/PB smear

• G-banded cytogenetic slides

• H &E stained slides

• PET section

• Each new probe/lot is evaluated with positive and negative controls to assay sensitivity and specificity and to determine the cut-off level.

• Negative and positive controls are tested with each probe hybridization with patient samples.

• At least two independent observers score for each assay (200 cells per observer).

FISH quality control

Each probe is tested at least on five normal controls of appropriate tissues. Statistical analysis: mean±3SD ---> cut-off level.

BCR/ABL-DF 0%

AML1/ETO-DF 0%

PML/RARA-DF 0%

MYC/IGH-DF 0%

BCL1/IGH-DF 0%

AML1/TEL-ES 9.4%

MLL-DC 2.2%

IGH-DC 2.6%

CBFb-DC 3.3%

CEP8 (gain) 1.94%

CEP12 (gain) 2.9%

(loss) 7.6%

Chr. 13 (loss) 2.8%

deletion of 13q14.3 9.1%

deletion of ATM/11q22.3 7.6%

deletion of TP53/17p13.1 8.6%

X/Y in male donor 0.8% XX

in female donor 0% XY

Probes cut-off Probes cut-off

Determination of cut-off level for positive results:

FISH: Dual fusion probe for t (8;14): IgH (14), MYC (8), Cen8 Aqu

nl 8

der(8)t(3;8;14)

nl 8

der(14)t(3;8;14)

der(3)t(3;8;14)

der(18)t(14;18)

der(14)t(14;18)

der(14)t(14;18)

der(3)t(3;8;14)

der(18)t(14;18)

der(14)t(3;8;14)

nl 18

FISH: Dual fusion probe for t (14;18): IgH (14), Bcl-2 (18)

nl 3

der(3)t(3;8;14)

der(8)t(3;8;14)

FISH: dual color break-apart probe for BCL-6 (3q27): 5’ red, 3’ green

Cytogenetics vs FISH: plus and minus

Cytogenetics

Plus: • Scan for abnormalities of all

chromosomes, arms, regions and bands of a cell.

• Diagnostic: specific chromosome abnormalities.

• Identify new tumor clone markers for follow-up.

• Clonal evolution evidences

Minus:• Needs fresh samples,• Need dividing cells and analyzable

metaphase cells.• Low sensitivity (1/20).• Low resolution (>10 Mb): missing

subtle and cryptical changes.• Heavily rely on technicians’

experience.

FISH

Plus:• Easier, simpler and faster. • High sensitivity (of 200 cells),

i.e., follow-up of RD.• High resolution(>100 kb).• Single cell analysis; Correlate with

morphology and immunophenotyping.

• no metaphase cells needed.

Fresh tissue or fixed section.

Terminally differentiated cells.

Low mitotic cells (CLL).

Minus:• Target regions only. • No whole chromosome pictures.• Limited probes: not many

commercial probes available.

indicated

• All diagnostic samples of leukemia and lymphoma (confirmed or suspicious).

• All evolving, transforming or relapsed samples.

• Residual disease samples if diagnostic samples are not analyzed.

• All follow-up samples at RD or CR if the diagnostic sample was abnormal in cytogenetic analysis.

• 1st sample after BMT for disease markers or polymorphisms.

Triaging cytogenetic/FISH analysis

NOT indicated

• All reactive or benign samples.• BM or PB with no involvement of NHL.• RD and CR samples if the diagnostic sample was normal (unless there

are changes in morphology/immunology).• Post-transplant samples with 100% donor cells (XX/XY) or remission

sample with no known chromosome abnormalities in FISH study.

Cytogenetics or FISH, or Both tests?

• All newly diagnosed AML/MDS cases need cytogenetics first:If specific chromosome abnormalities are known for certain subtype, and cytogenetic analysis is normal, FISH should be added.

if rush, FISH for specific chromosome abnormalities may be requested first.

• In RD cases with known chromosome abnormalities, such as t(9;22) in CML, either cytogenetics or FISH are needed. If cytogenetics is negative or inadequate, FISH will be helpful.

If cytogenetics is positive, FISH will not provide more information.

• At CR or MRD status, FISH is more helpful than cytogenetics in detection of the known chromosome abnormalities (if probes available).

Is ordering a FISH panel for AML, MDS, and NHL justified?

• Multiple comparison of conventional cytogenetic and FISH tests in several large series of AML and MDS in 1990s showed that additional common chromosome abnormalities is 2-4% by FISH using 7-8 probes in AML and MDS with complete (20 cells) cytogenetic analysis.

• FISH panel can detected common chromosome abnormalities in about 30% of AML and MDS with inadequate cytogenetic analysis.

Recommendations

•Cytogenetic analysis first in all newly diagnosed AML, MDS and MPN.•If cytogenetics is inadequate, FISH with panel is warranted in AML/MDS.•Once a chromosome abnormality is identified at DX, FISH is performed to follow up for disease status and treatment response.•FISH selectively detect recurring translocations in various subtypes of NHL.

NO

Application of genomic array analysis in leukemia and lymphoma---potentials and problems

Copy neutral LOH on chromosome 11

Discoveries: Genome-Wide Copy Number Analyses

Mullighan et al. Science 2008 322:1377

Genome-wide analysis of copy number changes in diagnosis ALL samples

Common clonal origin of relapse and diagnosis samples

1. 89% retained ≥1 diagnosis CNA at relapse2. 86% of pairs shared identical antigen receptor CNA at diagnosis and

relapse antigen receptors at diagnosis and relapse

Backtracking relapse-acquired CNA• Clonal evolution, or relapse clone present at low levels at diagnosis?• PCR assays for 10 relapse-acquired CNA: 7 present at diagnosis

Evolution of diagnosis and relapse clones

Potentials and problems of SNP array in leukemia and lymphoma

• High resolution;• No dividing cells;• Detect copy number alteration;• Detect LOH (deletion or partial UPDs);• Provide new insights of the genetic mechanisms of

leukemia/lymphoma;• Recurring lesions, such as deletion of PAX5 in ALL and with distinct

associations with different subtypes;

• No balance translocations, inversions or Sequence mutations;• Low sensitivity, 20-30% abnormal cells minimal;• Mosacisms and clonal evolution?• Primary or secondary changes?• Candidate genes in the critical regions of pUPDs/deletions?• Clinical significance? Survival, prognosis, subclassification, risk

grouping and treatment.

Can cytogenetics and FISH survive in the modern genomic era?

Thanks!

Next-generation sequencing

Common new CNA at diagnosis

Locus B TDeletionCDKN2A/B 16 2

ETV6 10 1IKZF1 5 2NR3C1 4 0TCF3 3 0DMD 2 0

ARPP-21 2 0BTLA/CD200

2 1

RAG1/2 2 0IKZF2 1 1GainMYB 0 2

CDKN2A/B

ETV6

The power of SNP analysis in ALL

• Genomic analyses provide new insights of the genetic mechanisms of ALL;

• Recurring lesions, such as deletion of PAX5, common in most subtypes of ALL and with distinct associations with different subtypes;

• IKZF1 alterations are a critical determinant of poor outcome;• Bioinformatics is critical to identify new therapeutic targets

based on SNP data;• Existing analysis limited: copy number alteration, gene

expression, limited sequencing;

Comparison of cytogenetics, FISH and SNP microarray

 Techniques  Cytogenetics   FISH  SNP microarray 

Resolution   +   ++   ++++  

Sensitivity   +   +++  + 

neutral LOH  –   - +   

cell division   +  –  –  

balanced lesions  +   + -  

multiple clones   + –/+   –  

Screening for unknown defects   +   - +