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
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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.
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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.
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
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
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.
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