Transcript ChgoPath-Zhang-1-9-12 - The Chicago Pathology Society

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 analysis
41-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
Acute myeloid leukemia
with t(8;21)(q22;q22)
94% of cells show a dualfusion signal pattern, i.e.
the AML1/ETO fusions
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
Cancer Cytogenetics
•
Samples:
(fresh!)
bone marrow (aspirate or core)
peripheral blood
lymph node/spleen/tonsil
solid tumor mass
CNS, plural fluids, etc
•
Culturing:
no mitogens added
short 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.
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(8;14)(q24.1;q32)
Burkitt Leuk./NHL
t(14;18)(q32;q21)
FL
t(11;14)(q13;q32)
MCL
t(8;21) in AML

Identified by Dr. Janet D. Rowley in 1973 as the first recurring
translocation in acute leukemia.

Associated with AML-M2 (~30% of AML-M2 cases, or ~5-10%
of all AML).

Characterized by a good response to therapy (98% CR) and a
prolonged disease-free survival.
Characteristic morphology:

myeloid blasts with indented nuclei.

basophilic cytoplasm with few
azurophilic granules.

increased eosinophils in bone
marrow.

Aberrant expression of CD19, and
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(3;21)
AML1-EVI1
Rare cases of
CML and MDS
t(12;21)
TEL-AML1
25% pediatric ALL
t(8;21)
AML1-ETO
10% AML
Point
Mutation
10%
t(16;21)
AML1-MTG16
rare cases of AML
inv(16)
CBF-MYH11
8% AML
AML1
CBF
Target genes
---TGTGGT--Core enhancer sequence
IL-3, GM-CSF
MPO, CSF-1R, TCR,
Consequences of chromosome translocations
1. Deregulated expression of a normal protein
t(8;14)
Increased
expression
of c-MYC
Promoter of IgH
Coding regions of c-MYC
2. Production of a fusion protein
t(8;21)
Coding regions of AML1
AML1-ETO
Coding regions of ETO
ETO-AML1
Expression
of a fusion
protein
AML1-ETO
Hematopoietic cell differentiation and chromosome
abnormalities in leukemia and lymphoma
ALL
AML
t(9;22) t(11q23)
t(11q23)
AML
t(8;21)
inv(16)
t(15;17)
Mast cell
Erythrocytes
Myeloid progenitor
hematopoietic
stem cell
Lympho-myeloid
Stem cell
Platelets
Lymphoid Progenitor
Eosinophil
CML
t(9;22)
ALL
t(12;21)
t(1;19)
t(8;14)
Hyperdipl.
Neutrophil
B cell
T cell
Monocyte
NHL
t(8;14), t(14;18)
t(11;14)
ALL/NHL
t(14q11.2)
t(7q34)
Recurring Chromosome Abnormalities in B-ALL
Immunophenotype
CD19
CD10
CD34
Ig M
Pro B
Common B
Pre B
B
+
+/-
+
+
+ (most)
-
+
+
+/+ (cytopl)
+
t(4;11)
t(12;21)
hyperdipl. (>50)
t(9;22)
t(12;21)
t(1;19)
hyperdipl.
t(8;14)
t(2;8)
t(8;22)
+ (surface)
Cytogenetic Pattern
Features of therapy-related AML
Alkylating agents
Radiation
Cytogenetics
Latency
Presentation
Prognosis
Topo II inhibitors
(VP16, Dox)
-5/del(5q)/-7/del(7q)
11q23, 21q22
5-7 yrs
2-3 yrs
Insidious (t-MDS)
acute
poor
poor
MRC/NCRI AML Trials: Overall Survival
ages 16-59, 2550 patients, 10 years follow-up
% alive
t(15;17), n=607
t(8;21), n=421
inv(16)/t(16;16),
n=284
t(9;11), n=61
t(3;5), n=25
t(6;9), n=42
AML/MDS, n=343
other 11q, n=60
t(9;22), n=44
-7/del(7q), n=336
-5/del(5q), n=258
Inv(3)/t(3;3),n=69
Years from entry
* Normal karyotypes: 38% OS
Grimwade et al., Blood, April 12, 2010
Clinical significance of chromosome abnormalities
in leukemia and lymphoma
•
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)
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, MFISH).
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
FISH test menu at NMH Cytogenetics Laboratory
AML
t(8;21) ETO/AML1
t(15;17) PML/RARα
Inv(16) CBFβ
11q23/MLL
17q21/RARα
inv(3)/t(3;3)
ALL
t(9;22) BCR/ABL
t(12;21) TEL/AML1
+4/+10/+17
del(9p)/p16/CEP9
11q23/MLL
MDS/MPN
del(5q)
del(7q)/-7
trisomy 8
del(13q)/-13
del(20q)
t(9;22) BCR/ABL
del(4q12)/CHIC2
CLL Panel
t(11;14), ATM/11q,
CEP12, del(13q),
del(17p), 14q32/IGH,
MYB/6q23
MM Panel
t(11;14), del(13q),
del(17p), 14q32/IGH
NHL
t(8;14) MYC/IGH
t(11;14)
CCND1/IGH
t(11;18)
API2/MALT1
t(14;18) IGH/BCL2
t(14;18) IGH/MALT1
3q27/BCL6
8q24/MYC
11q13/CCND1
14q32/IGH
18q21/BCL2
18q21/MALT1
2p23/ALK
ParaffinEmbedded
Tissue (PET)
HER2/CEP17
del(1p)/del(19q)
PTEN/CEP10
EGFR/CEP7
ALK
EWSR1
SS18
FOX01
BMT
X and Y chromosomes
Sample types and preparation for FISH
• Bone Marrow
•
Fresh BM/PB/LN
• Peripheral blood
•
Cytospin slides
• Lymph node
•
BM/PB smear
• Tumor mass
•
G-banded
cytogenetic slides
•
H &E stained slides
•
PET section
• CSF
• Plural fluid
FISH quality control
• 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).
Determination of cut-off level for positive results:
Each probe is tested at least on five normal controls of appropriate tissues.
Statistical analysis: mean±3SD ---> cut-off level.
Probes
cut-off
Probes
cut-off
BCR/ABL-DF
0%
CEP8 (gain)
1.94%
AML1/ETO-DF
0%
CEP12 (gain)
2.9%
PML/RARA-DF
0%
(loss)
7.6%
MYC/IGH-DF
0%
Chr. 13 (loss)
2.8%
BCL1/IGH-DF
0%
deletion of 13q14.3
9.1%
AML1/TEL-ES
9.4%
deletion of ATM/11q22.3
7.6%
MLL-DC
2.2%
deletion of TP53/17p13.1
8.6%
IGH-DC
2.6%
X/Y
in male donor
0.8% XX
CBFb-DC
3.3%
in female donor
0% XY
der(18)t(14;18)
nl 8
nl 8
der(14)t(3;8;14)
der(14)t(14;18)
der(3)t(3;8;14)
der(8)t(3;8;14)
FISH: Dual fusion probe for t (8;14): IgH (14), MYC (8), Cen8 Aqu
der(14)t(14;18)
der(3)t(3;8;14)
der(18)t(14;18)
nl 18
der(14)t(3;8;14)
FISH: Dual fusion probe for t (14;18): IgH (14), Bcl-2 (18)
der(8)t(3;8;14)
nl 3
der(3)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.
Triaging cytogenetic/FISH analysis
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.
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? NO
• 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.
Application of genomic array analysis
in leukemia and lymphoma
---potentials and problems
Copy neutral LOH on chromosome 11

This bone marrow sample has 55 Mb of copy neutral LOH on chromosome 11q.
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 relapse
2. 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?
Next-generation
sequencing
Thanks!
Common new CNA at diagnosis
CDKN2A/B
Locus
Deletion
B
T
CDKN2A/B
ETV6
IKZF1
NR3C1
TCF3
DMD
ARPP-21
BTLA/CD20
0
RAG1/2
IKZF2
16
10
5
4
3
2
2
2
1
2
0
0
0
0
2
1
2
1
0
1
0
2
Gain
MYB
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
+
-
+