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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.
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)
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)
• 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
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
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
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.
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;