Transcript Using PCR in Haematopathology

Using PCR in
Haematopathology
Paula Waits
Molecular Oncology
Bristol Genetics Laboratory
Introduction
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With the advent of improved methods for
extracting better quality DNA from both
paraffin embedded tissues (slides and curls) and
fresh tumour tissue, the use of PCR for
amplification of specific markers of disease has
become a useful diagnostic tool in
heamatopathology.
DNA Extraction
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In BGL we use a formalin fixed paraffin embedded
tissue kit or tumour DNA extraction kit (Qiagen) for
histopathology specimens and a routine DNA
extraction on our high throughput robot for peripheral
blood.
Sample requirements:
8 10um thick sections or 4 20um thick sections cut on a clean
microtome blade and send in a sterile Eppendorf tube.
Fresh snap frozen tumour sample on dry ice
One H+E slide marked clearly with the area of interest plus 10
serial unstained sections on slides
Peripheral blood or bone marrow (5-10 mls in EDTA)
DNA Extraction Cont…..
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Most molecular oncology PCR tests require between
20-100ng DNA dependent on the test eg a simple
JAK2V617F test for diagnosis of myeloproliferative
neoplasms requires 30ng of DNA in total while a
complex multiplex PCR for clonality in lymphoma
requires 100ng/ul per multiplex (14 in total) in
duplicate which can mean over 1mg for the whole test
to be completed!!!!
If we don’t receive the correct amount of sample we
may not be able to perform all of the tests required and
the quality of the results may be affected by inadequate
quantity/quality DNA.
Quality Checking
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The quality and quantity of a DNA sample is
identified by assessing the 230/260 and 260/280
ratio using the nanadrop (spectrophotometry)
Good Quality DNA
Poorer Quality DNA
Specimen control ladder
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It is also important that we run a specimen
control ladder with PET and tumour samples to
ensure that the DNA extracted is not too
fragmented as this can give false negative results.
Clonality Testing in
Lymphoma
Introduction
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The majority of lymphoid malignancies belong to the B
cell lineage (90-95%) with only 5-7% being T cells
According to van Dongen et al., (2003) the vast
majority of lymphoid malignancies (>98%) contain
identically (clonal) rearranged immunoglobulin (Ig)
and/or T cell receptor (TCR) genes.
Ig and TCR gene loci contain many different variable
(V), diversity (D) and joining (J) regions. In Ig heavy
chains (IGH), TCRB and TCRD there is an initial D-J
rearrangement followed by a V to D-J rearrangement
whereas there is only direct V-J rearrangements in IGK,
IGL, TCRA and TCRG genes.
Introduction cont….
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During the rearrangements of V, D and J gene
segments random deletions and insertions occur
at the junction sites.
This results in highly diverse junctional regions,
which in turn, leads to an Ig and TCR repertoire
of ~10(12).
IGH gene rearrangement
V genes
(124)
VH1
VH2
VH3
VH4 VH5
D genes
(27)
VHn
J genes
(6)
JH1 - 6
DH1 DH2 DH3 DHn
Constant region
Cm
Germline IgH locus
Partial DH-JH
rearrangement
Full VH-DH-JH
rearrangement
V
N
D
N
J
CDR3
Random insertion and deletion of nucleotides
Cd
Cg
Ca
Introduction cont….
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In mature B cells, somatic hypermutation of the
V(D)J exon of IgH and Ig light chain genes
occurs.
This causes single nucleotide mutations or
insertions/deletions to occur.
As such, mature B cell malignancies can show a
mutated or unmutated gene profile.
Important points to remember when
testing for clonality
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Clonality does not always imply malignancy – all
results must be interpreted in the context of all of the
other available diagnostic criteria.
Ig and TCR gene rearrangements are not markers
for lineage – Ig and TCR gene rearrangements are not
necessarily restricted to B and T cell lineages,
respectively. Cross lineage can occur ie a B cell
malignancy can be positive for a TCR gene
rearrangement and vice versa.
Limited sensitivity compared to polyclonal
background – clonality can only be reported if the
clonal peak is 3x higher than the 3rd highest peak in the
polyclonal background
BGL and Clonality Testing using IdentiClone gene
clonality assay kits from InVivoScribe Technologies
DNA sample
Liu et al.,
(2007)
DNA size ladder PCR if paraffin-embedded tissue
(If DNA > 300 bp)
B-cell proliferation
IGHB + IGKA+B
91% (58%)
T-cell proliferation
Initial Screen
If not clonal
IGHA+C+D
99% (79%)
IGL + IGHE
100% (80%)
Extended Screen
If not clonal
If not clonal
TCRGA+B
94% (30%)
T-cell lineage
unknown
TCRBA+B
98% (73%)
TCRαβ+
TCRBA+B
+C
TCRBC + TCRD
100% (82%)
No evidence of presence of clonal
B/T-cell population in the sample by PCR
TCRγδ+
TCRBC
+ TCRD
Patient A
Clinical
 Sample type: Paraffin embedded tissue
 Referred with extensive mediastinal and abdominal
lymphadenopathy. Diagnosis hypercalcaemia ?lymphoma
 Immunology: A small population of large B cells with the
phenotype CD19, CD20 and CD79+, strong Kappa+, CD10, 5,
103 Cell Pathology Summary: Histology suggestive of diffuse large B
cell lymphoma although check immunophenotype and genetic
analysis by PCR requested for confirmation.
 Immunohistochemistry: Tumour cells +ve with CD20 and
BCL2. +ve nuclear staining with BCL6, MUM1,PAX5 and p53
Patient A Clonality Testing
IGH Tube B VH(1-7) FR2-JH
G B C .0 9 .5 6 5 9 IG H B L Y M P H O M A P C R 0 6 .C 1 0 _ 0 9 0 7 2 2 1 7 A V
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Dye Signal
Clonal peak
identified at
271bp
6
5
4
3
2
1
0
0
0
0
0
0
0
271.59
2 2 5
+ V E I G H B 2 L5 0 Y M P H O M A2 7 S 5 i Pz e C ( n Rt ) 0 6 . F 1 0 3 _ 0 00 9 0 7 2 2 1 7 A Z3 2 5
3 5 0
1 2 5 0 0 0
+ve
Control
285.57
1 0 0 0 0 0
7 5 0 0 0
5 0 0 0 0
2 5 0 0 0
Dye Signal
0
2 2 5
-V E IG H
1 2 5
1 0 0
7 5
5 0
2 5
0
0
0
0
0
2 5 0
B L Y M P H O M A
2 7 5
S iz e (n t)
3 0 0
3 2 5
3 5 0
P C R 0 6 .G 1 0 _ 0 9 0 7 2 2 1 7 B 0
Dye Signal
0
0
0
0
0
0
Polyclonal
control
2 2 5
2 5 0
2 7 5
S iz e (n t)
3 0 0
3 2 5
Final Diagnosis: Diffuse Large B Cell Lymphoma
3 5 0
Patient B
Clinical
 Sample Type: Tumour
 Referred with ? Lymphoma. Lymphadenopathy left
neck
 Very difficult lymph node to interpret histologically
 Immunohistochemistry: CD10, Cyclin D1 and bcl2
negative
 Working diagnosis: Marginal zone lymphoma with
follicular colonisation.
Patient B Clonality Testing
IGH Tube B VH(1-7) FR2-JH
L W
C .0 9 .1 0 4 1 3 IG H B L Y M P H O M A P C R 2 7 .A 0 6 _ 0 9 1 2 2 3 1 0 9 L
1 0 0 0 0
7 5 0 0
5 0 0 0
254.73
2 5 0 0
0
Dye Signal
3rd largest peak in
polyclonal background
~1000, clonal peak
~4000 therefore clonal
peak over 3x the
height of the
polyclonal background
and reported as weakly
clonal in a polyclonal
background
2 3 0
2 4 0
2 5 0
+ V E IG H
2 6 0
B
L Y M P H O M A
7 5 0 0 0
2 7 0
S iz e (n t)
2 8 0
2 9 0
3 0 0
3 1 0
P C R 2 7 .C 0 6 _ 0 9 1 2 2 3 1 0 9 N
+ve
Control
260.84
5 0 0 0 0
2 5 0 0 0
Dye Signal
0
2 3 0
2 4 0
2 5 0
-V E IG H
1 2 5
1 0 0
7 5
5 0
2 5
0
0
0
0
0
2 6 0
B L Y M P H O M A
2 7 0
S iz e (n t)
2 8 0
2 9 0
3 0 0
3 1 0
P C R 0 6 .G 1 0 _ 0 9 0 7 2 2 1 7 B 0
Dye Signal
0
0
0
0
0
0
Polyclonal
Control
2 2 5
2 5 0
2 7 5
S iz e (n t)
3 0 0
Final Diagnosis: Nodal marginal zone lymphoma
3 2 5
3 5 0
Patient C
Clinical
 Sample: PET
 Rt groin biopsy ? Lymphoma – anaemic axillary, inguinal and
para-aortic lymphodenopathy – Urgent
 Initial histology: reactive hyperplasia but sent to lymphoma
expert for opinion who then sent a PET section to BGL for B
and T cell clonality testing
 Concurrently, a peripheral blood sample was sent for JAK2
V617F testing – no mutation present.
 Immunology showed a 1-2% population of Lambda +ve cells
 Initial B and T cell screen on PET = no clonality identified =
reactive rather than malignant
 Further testing, an initial B and T cell screen on peripheral blood
showed……….
Patient C Clonality Testing (PET
and Peripheral blood)
TCRG (tube A) VγIf Vγ10 –
Jγ1.3/2.3 + Jγ1.1/2.1
B J C 1 0 .2 3 5
0
0
0
0
0
0
0
0
0
0
0
0
T C R G
T U B E
A
P C R 3 0
L Y M
P H O M
A .E 0 4 _ 1 0 0 1 1 4 1 1 Y W
0
0
0
0
0
0
0
PET
1 7 0
B L
1 8 0
C 1 0 .6 6 5
1 9 0
T C R G
2 0 0
T U B E
2 1 0
A
P C R
2 2 0
2 3 0
S iz e (n t)
3 4
L Y M
P H O M
1 0 0 0 0 0
2 4 0
2 5 0
2 6 0
A .F 0 3 _ 1 0 0 1 2 8 0 8 4 8
241.80
240.91
7 5 0 0 0
PB
5 0 0 0 0
2 5 0 0 0
0
Dye Signal
3rd largest peak in polyclonal
background ~25000, clonal
peak ~75000 therefore clonal
peak over 3x the height of the
polyclonal background and
reported as weakly clonal in a
polyclonal background
0
5
0
5
0
5
Dye Signal
Negative for the detection of
clonal TCRG chain
rearrangements
3
2
2
1
1
1 5 0
1 7 5
2 0 0
2 2 5
2 5 0
2 7 5
S iz e (n t)
+ V
1 5 0 0
1 2 5 0
1 0 0 0
7 5 0
5 0 0
2 5 0
E
T C R G
T U
B E
A
P C R
3 4
Dye Signal
0
0
0
0
0
0
0
L Y
M
O
M
A
. H
0 3 _ 1 0 0 1 2 8 0 8 4 B
211.69
1 5 0
1 7 5
+VE
2 0 0
2 2 5
S iz e
- V
2 0 0 0
1 5 0 0
1 0 0 0
5 0 0
P H
E
T C R G
T U
B E
A
P C R
3 4
L Y
M
2 5 0
2 7 5
(n t)
P H
O
M
A
.A
0 4 _ 1 0 0 1 2 8 0 8 4 B
Dye Signal
0
0
0
0
0
PC
1 5 0
1 7 5
2 0 0
2 2 5
S iz e
(n t)
2 5 0
2 7 5
What went wrong with the clonality
testing?
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Nothing!!
We reported that in the context of overall diagnostic criteria,
clonal cell populations can indicate the presence of
haematological malignancy.
While monoclonality is a key feature of tumour cell populations
it does not always imply malignancy because some reactive
processes contain large clonal lymphocyte
populations. Therefore we need to take into consideration the
whole clinical picture rather than just the result of the clonality
testing.
The patients flow results showed a 1-2% population of
malignant cells which were Lambda +ve, this is part of the
extended panel which also includes other B cell markers
(Framework 1 and 3 and the incomplete DH’s)
However, its absence would not detract from the clonality
already identified in the T cells
Patient C Cont….
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An extended B cell screen was initiated and was
negative for the detection of IGH rearrangements
A second core biopsy was taken. The histology showed
HHV8 (human herpes virus-8) positive Kaposi’s
sarcoma and HHV8 associated lymphoproliferative
disorder. HIV negative patient, probably common
variable immunodeficiency.
HHV8 positive plasma cells are monotypic but
polyclonal. (On review first biopsy also HHV8+ but
not Castlemans Disease))
Patient now treated with Rituximab (anti-CD20
monoclonal antibody therapy) and responding well
Other PCR Techniques
used in HaematoOncology
Allele Specific PCR
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Amplification of specific alleles, or DNA sequence
variants, at the same locus. Specificity is achieved by
designing one or both PCR primers so that they
partially overlap the site of sequence difference
between the amplified alleles.
Used in haemato-oncology for detection of
JAK2V617F mutation in myleoproliferative neoplasms.
GGA GTA TGT GTC T (WT) Pos = White Neg = Yellow
GGA GTA TGT TTC T (Mut)
Capillary Electrophoresis
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Gel-capillary electrophoresis is an alternative to
traditional electrophoretic gel based techniques.
Many advantages including excellent resolution which
enables multiplex reactions (ie more than one set of
primers in each reaction mix) reduced sample
quantities, and automation.
Used as the detection method for many molecular
oncology tests including clonality testing in lymphoma
and Loss of heterozygosity in oligodendrogliomas
Blood
Tumour
Real-time quantitative PCR (RQ-PCR)
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Quantitative real-time polymerase chain reaction (PCR) provides
an accurate method for determination of levels of specific DNA
and RNA (RT) sequences in tissue samples. It is based on
detection of a fluorescent signal produced proportionally during
amplification of a PCR product.
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Used for quantification of BCR-ABL molecules in chronic
myeloid leukaemia (left) and minimal residual disease detection
in acute lymphoblastic leukaemia (right)
Logarithmic
dilution series
Positive
Control
10(6)
10(5)
10(3)
10(2)
10(1)
test
Day 28
samples
Sequencing
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DNA sequencing is the process of determining the
nucleotide order of a given DNA fragment.
Most automated laboratories use in dye-terminator
sequencing, where each of the four dideoxynucleotide
chain terminators (A,C,G,T) is labelled with a
fluorescent dye with a different wavelength and is
compared to a wild type sequence for identification of
changes in the sequence.
4bp inserted
duplication
in the NPM1
gene in AML
Pyrosequencing
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High throughput method of DNA sequencing based on a
“sequence by synthesis” principle. In short, this method
synthesizes the complementary strand of a single strand of
DNA one base pair at a time.
When the complementary base binds to the single stranded
DNA a chemiluminescent signal is given off which can be
detected, allowing the determination of the sequence in real
time.
In BGL this technology will be developed to allow for screening
of mutations associated with myeloproliferative neoplasms (for
example JAK2V617F and MPLW515K/L), K-Ras testing in
colorectal carcinoma and methylation status in glioblastoma
brain tumours.
High resolution melt analysis
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Perform a PCR using primers specific for gene in
question
Following the PCR, heat the amplicon DNA from
around 50˚C up to around 95˚C.
At some point during this process, the melting
temperature of the amplicon is reached and the two
strands of DNA separate or “melt” apart
The dsDNA is labelled with a fluorescent dye, which
fluoresces brightly. As the stands separate the intensity
of the dye is reduced and this reduction is measured in
real time producing a melt curve
High resolution melt analysis cont..
http://en.wikipedia.org/wiki/High
_Resolution_Melt
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BGL is currently developing this technology for testing
of MPL and JAK2 Exon 12 mutations in MPNs but
many papers have used it for BRCA1 and BRCA2
mutation screening and TP53 screening in breast and
ovarian cancer which we are ?hoping to develop in the
future.
References
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Van Dongen et al., Design and standardisation of PCR
primers and protocols for detection of clonal
immunoglobulin and T cell receptor gene
recombinations in suspect lymphoproliferations.
Leukemia, 2003. 17: 2257-2317
Liu H et al., A practical study for the routine use of
BIOMED-2 PCR assays for the detection of B and T
cell clonality in diagnostic haematopathology. Br J
Haematol. 2007. 138(1):31-43
Acknowledgements
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Eileen Roberts (Head of Department, BGL)
Dr Jerry Hancock (Molecular Oncology, BGL)
Helen, Rich, Paul, Jess and Adelea (Molecular
Oncology team)
Dr Nicholas Rooney (Cellular Pathology)
Konstantin Sidelnikov (InVivoScribe
Technologies, Inc)