Using PCR in Using PCR in HaematopatholoHaematopatholo

gygyPaula WaitsPaula Waits

Molecular OncologyMolecular Oncology

Bristol Genetics LaboratoryBristol Genetics Laboratory

IntroductionIntroduction

With the advent of improved With the advent of improved methods for extracting better quality methods for extracting better quality DNA from both paraffin embedded DNA from both paraffin embedded tissues (slides and curls) and fresh tissues (slides and curls) and fresh tumour tissue, the use of PCR for tumour tissue, the use of PCR for amplification of specific markers of amplification of specific markers of disease has become a useful disease has become a useful diagnostic tool in heamatopathology.diagnostic tool in heamatopathology.

DNA ExtractionDNA Extraction In BGL we use a formalin fixed paraffin In BGL we use a formalin fixed paraffin

embedded tissue kit or tumour DNA embedded tissue kit or tumour DNA extraction kit (Qiagen) for histopathology extraction kit (Qiagen) for histopathology specimens and a routine DNA extraction on specimens and a routine DNA extraction on our high throughput robot for peripheral our high throughput robot for peripheral blood.blood.

Sample requirements: Sample requirements: 8 10um thick sections or 4 20um thick sections cut on 8 10um thick sections or 4 20um thick sections cut on

a clean microtome blade and send in a sterile a clean microtome blade and send in a sterile Eppendorf tube.Eppendorf tube.

Fresh snap frozen tumour sample on dry iceFresh snap frozen tumour sample on dry ice One H+E slide marked clearly with the area of One H+E slide marked clearly with the area of

interest plus 10 serial unstained sectionsinterest plus 10 serial unstained sections on slideson slides Peripheral blood or bone marrow (5-10 mls in EDTA)Peripheral blood or bone marrow (5-10 mls in EDTA)

DNA Extraction Cont…..DNA Extraction Cont….. Most molecular oncology PCR tests require Most molecular oncology PCR tests require

between 20-100ng DNA dependent on the test between 20-100ng DNA dependent on the test eg a simple JAK2V617F test for diagnosis of eg a simple JAK2V617F test for diagnosis of myeloproliferative neoplasms requires 30ng myeloproliferative neoplasms requires 30ng of DNA in total while a complex multiplex of DNA in total while a complex multiplex PCR for clonality in lymphoma requires PCR for clonality in lymphoma requires 100ng/ul per multiplex (14 in total) in 100ng/ul per multiplex (14 in total) in duplicate which can mean over 1mg for the duplicate which can mean over 1mg for the whole test to be completed!!!!whole test to be completed!!!!

If we don’t receive the correct amount of If we don’t receive the correct amount of sample we may not be able to perform all of sample we may not be able to perform all of the tests required and the quality of the the tests required and the quality of the results may be affected by inadequate results may be affected by inadequate quantity/quality DNA.quantity/quality DNA.

Quality CheckingQuality Checking The quality and quantity of a DNA The quality and quantity of a DNA

sample is identified by assessing the sample is identified by assessing the 230/260 and 260/280 ratio using the 230/260 and 260/280 ratio using the nanadrop (spectrophotometry)nanadrop (spectrophotometry)

Good Quality DNA Poorer Quality DNA

Specimen control ladderSpecimen control ladder It is also important that we run a It is also important that we run a

specimen control ladder with PET specimen control ladder with PET and tumour samples to ensure that and tumour samples to ensure that the DNA extracted is not too the DNA extracted is not too fragmented as this can give false fragmented as this can give false negative results.negative results.

Clonality Testing Clonality Testing in Lymphomain Lymphoma

IntroductionIntroduction The majority of lymphoid malignancies belong to The majority of lymphoid malignancies belong to

the B cell lineage (90-95%) with only 5-7% being the B cell lineage (90-95%) with only 5-7% being T cellsT cells

According to van Dongen et al., (2003) the vast According to van Dongen et al., (2003) the vast majority of lymphoid malignancies (>98%) majority of lymphoid malignancies (>98%) contain identically (clonal) rearranged contain identically (clonal) rearranged immunoglobulin (Ig) and/or T cell receptor (TCR) immunoglobulin (Ig) and/or T cell receptor (TCR) genes.genes.

Ig and TCR gene loci contain many different Ig and TCR gene loci contain many different variable (V), diversity (D) and joining (J) regions. variable (V), diversity (D) and joining (J) regions. In Ig heavy chains (IGH), TCRB and TCRD there In Ig heavy chains (IGH), TCRB and TCRD there is an initial D-J rearrangement followed by a V to is an initial D-J rearrangement followed by a V to D-J rearrangement whereas there is only direct D-J rearrangement whereas there is only direct V-J rearrangements in IGK, IGL, TCRA and V-J rearrangements in IGK, IGL, TCRA and TCRG genes.TCRG genes.

Introduction cont….Introduction cont….

During the rearrangements of V, D During the rearrangements of V, D and J gene segments random and J gene segments random deletions and insertions occur at the deletions and insertions occur at the junction sites.junction sites.

This results in highly diverse This results in highly diverse junctional regions, which in turn, junctional regions, which in turn, leads to an Ig and TCR repertoire of leads to an Ig and TCR repertoire of ~10(12).~10(12).

IGH gene IGH gene rearrangementrearrangement

Partial DH-JH rearrangement

Full VH-DH-JH rearrangement

V DN JN

CDR3

Random insertion and deletion of nucleotides

D genes(27)

J genes(6)

V genes(124)

Constant region

Germline IgH locus

VH1 VH2 VH3 VH4 VHn DH1 DH2 DH3 DHn JH1 - 6 Cm Cd Cg CaVH5

Introduction cont….Introduction cont…. In mature B cells, somatic In mature B cells, somatic

hypermutation of the V(D)J exon of IgH hypermutation of the V(D)J exon of IgH and Ig light chain genes occurs.and Ig light chain genes occurs.

This causes single nucleotide mutations This causes single nucleotide mutations or insertions/deletions to occur. or insertions/deletions to occur.

As such, mature B cell malignancies As such, mature B cell malignancies can show a mutated or unmutated gene can show a mutated or unmutated gene profile.profile.

Important points to Important points to remember when testing for remember when testing for

clonalityclonality Clonality does not always imply Clonality does not always imply

malignancymalignancy – all results must be interpreted – all results must be interpreted in the context of all of the other available in the context of all of the other available diagnostic criteria.diagnostic criteria.

Ig and TCR gene rearrangements are not Ig and TCR gene rearrangements are not markers for lineagemarkers for lineage – Ig and TCR gene – Ig and TCR gene rearrangements are not necessarily restricted rearrangements are not necessarily restricted to B and T cell lineages, respectively. Cross to B and T cell lineages, respectively. Cross lineage can occur ie a B cell malignancy can lineage can occur ie a B cell malignancy can be positive for a TCR gene rearrangement and be positive for a TCR gene rearrangement and vice versa.vice versa.

Limited sensitivity compared to polyclonal Limited sensitivity compared to polyclonal backgroundbackground – clonality can only be reported – clonality can only be reported if the clonal peak is 3x higher than the 3if the clonal peak is 3x higher than the 3rdrd highest peak in the polyclonal backgroundhighest peak in the polyclonal background

BGL and Clonality Testing using BGL and Clonality Testing using IdentiClone gene clonality assay kits IdentiClone gene clonality assay kits

from InVivoScribe Technologiesfrom InVivoScribe TechnologiesDNA sample

DNA size ladder PCR if paraffin-embedded tissue

(If DNA > 300 bp)

B-cell proliferation T-cell proliferationIGHB + IGKA+B91% (58%)

TCRGA+B94% (30%)

If not clonal

IGHA+C+D99% (79%)

IGL + IGHE100% (80%)

T-cell lineageunknown

TCRBA+B98% (73%)

TCRBA+B+C

TCRBC + TCRD

TCRBC + TCRD100% (82%)

If not clonal

No evidence of presence of clonalB/T-cell population in the sample by PCR

If not clonal

TCRαβ+TCRγδ

+

Initial Screen

Extended Screen

Liu et al., (2007)

Patient APatient AClinical Clinical Sample type: Paraffin embedded tissueSample type: Paraffin embedded tissue Referred with extensive mediastinal and abdominal Referred with extensive mediastinal and abdominal

lymphadenopathy. Diagnosis hypercalcaemia ?lymphadenopathy. Diagnosis hypercalcaemia ?lymphomalymphoma

Immunology: A small population of large B cells with Immunology: A small population of large B cells with the phenotype CD19, CD20 and CD79+, strong the phenotype CD19, CD20 and CD79+, strong Kappa+, CD10, 5, 103-Kappa+, CD10, 5, 103-

Cell Pathology Summary: Histology suggestive of Cell Pathology Summary: Histology suggestive of diffuse large B cell lymphoma although check diffuse large B cell lymphoma although check immunophenotype and genetic analysis by PCR immunophenotype and genetic analysis by PCR requested for confirmation.requested for confirmation.

Immunohistochemistry: Tumour cells +ve with CD20 Immunohistochemistry: Tumour cells +ve with CD20 and BCL2. +ve nuclear staining with BCL6, and BCL2. +ve nuclear staining with BCL6, MUM1,PAX5 and p53MUM1,PAX5 and p53

Patient A Clonality Patient A Clonality TestingTesting

0100002000030000400005000060000

225 250 275 300 325 350

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

Size (nt)

Dye

Sig

nal

271.59

0

25000

50000

75000

100000

125000

225 250 275 300 325 350

+ V E IG H B L Y M P H O M A P C R 0 6 .F 1 0 _ 0 9 0 7 2 2 1 7 A Z

Size (nt)

Dye

Sig

nal

285.57

0

2500

5000

7500

10000

12500

225 250 275 300 325 350

-V E IG H B L Y M P H O M A P C R 0 6 .G 1 0 _ 0 9 0 7 2 2 1 7 B 0

Size (nt)

Dye

Sig

nal

Final Diagnosis: Diffuse Large B Cell Lymphoma

+ve Control

Polyclonal control

Clonal peak identified at 271bp

IGH Tube B VH(1-7) FR2-JH

Patient BPatient B

ClinicalClinical Sample Type: TumourSample Type: Tumour Referred with ? Lymphoma. Referred with ? Lymphoma.

Lymphadenopathy left neckLymphadenopathy left neck Very difficult lymph node to interpret Very difficult lymph node to interpret

histologically histologically Immunohistochemistry: CD10, Cyclin D1 Immunohistochemistry: CD10, Cyclin D1

and bcl2 negativeand bcl2 negative Working diagnosis: Marginal zone Working diagnosis: Marginal zone

lymphoma with follicular colonisation.lymphoma with follicular colonisation.

Patient B Clonality Patient B Clonality TestingTesting

0

2500

5000

7500

10000

230 240 250 260 270 280 290 300 310

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

Size (nt)

Dye

Sig

nal 254.73

0

25000

50000

75000

230 240 250 260 270 280 290 300 310

+ V E IG H B L Y M P H O M A P C R 2 7 .C 0 6 _ 0 9 1 2 2 3 1 0 9 N

Size (nt)

Dye S

ignal

260.84

0

2500

5000

7500

10000

12500

225 250 275 300 325 350

-V E IG H B L Y M P H O M A P C R 0 6 .G 1 0 _ 0 9 0 7 2 2 1 7 B 0

Size (nt)

Dye

Sig

nal

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

Final Diagnosis: Nodal marginal zone lymphoma

+ve Control

Polyclonal Control

IGH Tube B VH(1-7) FR2-JH

Patient CPatient CClinicalClinical Sample: PET Sample: PET Rt groin biopsy ? Lymphoma – anaemic axillary, Rt groin biopsy ? Lymphoma – anaemic axillary,

inguinal and para-aortic lymphodenopathy – Urgentinguinal and para-aortic lymphodenopathy – Urgent Initial histology: reactive hyperplasia but sent to Initial histology: reactive hyperplasia but sent to

lymphoma expert for opinion who then sent a PET lymphoma expert for opinion who then sent a PET section to BGL for B and T cell clonality testingsection to BGL for B and T cell clonality testing

Concurrently, a peripheral blood sample was sent Concurrently, a peripheral blood sample was sent for JAK2 V617F testing – no mutation present.for JAK2 V617F testing – no mutation present.

Immunology showed a 1-2% population of Lambda Immunology showed a 1-2% population of Lambda +ve cells+ve cells

Initial B and T cell screen on PET = no clonality Initial B and T cell screen on PET = no clonality identified = reactive rather than malignantidentified = reactive rather than malignant

Further testing, an initial B and T cell screen on Further testing, an initial B and T cell screen on peripheral blood showed……….peripheral blood showed……….

Patient C Clonality Testing Patient C Clonality Testing (PET and Peripheral blood)(PET and Peripheral blood)

0

25000

50000

75000

100000

150 175 200 225 250 275

B L C 1 0 .6 6 5 T C R G T U B E A P C R 3 4 L Y M P H O M A .F 0 3 _ 1 0 0 1 2 8 0 8 4 8

Size (nt)

Dye S

ignal

240.91

241.80

0250050007500

100001250015000

150 175 200 225 250 275

+ V E T C R G T U B E A P C R 3 4 L Y M P H O M A .H 0 3 _ 1 0 0 1 2 8 0 8 4 B

Size (nt)

Dye S

ignal

211.69

0

5000

10000

15000

20000

150 175 200 225 250 275

-V E T C R G T U B E A P C R 3 4 L Y M P H O M A .A 0 4 _ 1 0 0 1 2 8 0 8 4 B

Size (nt)

Dye S

ignal

TCRG (tube A) VγIf Vγ10 – Jγ1.3/2.3 + Jγ1.1/2.1

05000

1000015000200002500030000

170 180 190 200 210 220 230 240 250 260

B J C 1 0 .2 3 5 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

Size (nt)

Dye S

ignal PE

T

PB

+VE

PC

Negative for the detection of clonal TCRG chain rearrangements

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

What went wrong with the What went wrong with the clonality testing?clonality testing?

Nothing!!Nothing!! We reported that in the context of overall diagnostic We reported that in the context of overall diagnostic

criteria, clonal cell populations can indicate the criteria, clonal cell populations can indicate the presence of haematological malignancy.  presence of haematological malignancy.  

While monoclonality is a key feature of tumour cell While monoclonality is a key feature of tumour cell populations it does not always imply malignancy populations it does not always imply malignancy because some reactive processes contain large because some reactive processes contain large clonal lymphocyte populations.  Therefore we need clonal lymphocyte populations.  Therefore we need to take into consideration the whole clinical picture to take into consideration the whole clinical picture rather than just the result of the clonality testing. rather than just the result of the clonality testing. 

The patients flow results showed a 1-2% population The patients flow results showed a 1-2% population of malignant cells which were Lambda +ve, this is of malignant cells which were Lambda +ve, this is part of the extended panel which also includes part of the extended panel which also includes other B cell markers (Framework 1 and 3 and the other B cell markers (Framework 1 and 3 and the incomplete DH’s) incomplete DH’s)

However, its absence would not detract from the However, its absence would not detract from the clonality already identified in the T cellsclonality already identified in the T cells

Patient C Cont….Patient C Cont…. An extended B cell screen was initiated and was An extended B cell screen was initiated and was

negative for the detection of IGH rearrangementsnegative for the detection of IGH rearrangements A second core biopsy was taken. The histology A second core biopsy was taken. The histology

showed HHV8 (human herpes virus-8) positive showed HHV8 (human herpes virus-8) positive Kaposi’s sarcoma and HHV8 associated Kaposi’s sarcoma and HHV8 associated lymphoproliferative disorder. HIV negative lymphoproliferative disorder. HIV negative patient, probably common variable patient, probably common variable immunodeficiency.immunodeficiency.

HHV8 positive plasma cells are monotypic but HHV8 positive plasma cells are monotypic but polyclonal. (On review first biopsy also HHV8+ polyclonal. (On review first biopsy also HHV8+ but not Castlemans Disease))but not Castlemans Disease))

Patient now treated with Rituximab (anti-CD20 Patient now treated with Rituximab (anti-CD20 monoclonal antibody therapy) and responding monoclonal antibody therapy) and responding wellwell

Other PCR Other PCR Techniques used in Techniques used in Haemato-OncologyHaemato-Oncology

Allele Specific PCRAllele Specific PCR Amplification of specific alleles, or DNA Amplification of specific alleles, or DNA

sequence variants, at the same locus. sequence variants, at the same locus. Specificity is achieved by designing one or Specificity is achieved by designing one or both PCR primers so that they partially both PCR primers so that they partially overlap the site of sequence difference overlap the site of sequence difference between the amplified alleles. between the amplified alleles.

Used in haemato-oncology for detection of Used in haemato-oncology for detection of JAK2V617F mutation in myleoproliferative JAK2V617F mutation in myleoproliferative neoplasms.neoplasms.

GGA GTA TGT GGA GTA TGT GTC GTC T (WT)T (WT) GGA GTA TGT GGA GTA TGT TTC TTC T (Mut)T (Mut)

Pos = White Neg = Yellow

Capillary ElectrophoresisCapillary Electrophoresis Gel-capillary electrophoresis is an Gel-capillary electrophoresis is an

alternative to traditional electrophoretic alternative to traditional electrophoretic gel based techniques.gel based techniques.

Many advantages including excellent Many advantages including excellent resolution which enables multiplex resolution which enables multiplex reactions (ie more than one set of primers reactions (ie more than one set of primers in each reaction mix) reduced sample in each reaction mix) reduced sample quantities, and automation. quantities, and automation.

Used as the detection method for many Used as the detection method for many molecular oncology tests including molecular oncology tests including clonality testing in lymphoma and Loss of clonality testing in lymphoma and Loss of heterozygosity in oligodendrogliomasheterozygosity in oligodendrogliomas

Blood

Tumour

Real-time quantitative PCR Real-time quantitative PCR (RQ-PCR)(RQ-PCR)

Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain reaction (PCR) provides an accurate method for (PCR) provides an accurate method for determination of levels of specific DNA and RNA determination of levels of specific DNA and RNA (RT) sequences in tissue samples. It is based on (RT) sequences in tissue samples. It is based on detection of a fluorescent signal produced detection of a fluorescent signal produced proportionally during amplification of a PCR proportionally during amplification of a PCR

product.product. UsedUsed for quantification of BCR-ABL molecules in for quantification of BCR-ABL molecules in

chronic myeloid leukaemia (left) and minimal chronic myeloid leukaemia (left) and minimal residual disease detection in acute lymphoblastic residual disease detection in acute lymphoblastic leukaemia (right)leukaemia (right)

testPositive Control10(6)

10(5)

10(3)

10(2)

10(1)

Day 28 Day 28 samplessamples

Logarithmic Logarithmic dilution seriesdilution series

SequencingSequencing DNA sequencing is the process of DNA sequencing is the process of

determining the nucleotide order of a determining the nucleotide order of a given DNA fragment. given DNA fragment.

Most automated laboratories use in dye-Most automated laboratories use in dye-terminator sequencing, where each of the terminator sequencing, where each of the four dideoxynucleotide chain terminators four dideoxynucleotide chain terminators (A,C,G,T) is labelled with a fluorescent (A,C,G,T) is labelled with a fluorescent dye with a different wavelength and is dye with a different wavelength and is compared to a wild type sequence for compared to a wild type sequence for identification of changes in the sequence.identification of changes in the sequence.4bp

inserted duplication in the NPM1 gene in AML

PyrosequencingPyrosequencing High throughput method of DNA sequencing based High throughput method of DNA sequencing based

on a “sequence by synthesis” principle. In short, this on a “sequence by synthesis” principle. In short, this method synthesizes the complementary strand of a method synthesizes the complementary strand of a single strand of DNA one base pair at a time. single strand of DNA one base pair at a time.

When the complementary base binds to the single When the complementary base binds to the single stranded DNA a chemiluminescent signal is given off stranded DNA a chemiluminescent signal is given off which can be detected, allowing the determination of which can be detected, allowing the determination of the sequence in real time.the sequence in real time.

In BGL this technology will be developed to allow for In BGL this technology will be developed to allow for screening of mutations associated with screening of mutations associated with myeloproliferative neoplasms (for example myeloproliferative neoplasms (for example JAK2V617F and MPLW515K/L), K-Ras testing in JAK2V617F and MPLW515K/L), K-Ras testing in colorectal carcinoma and methylation status in colorectal carcinoma and methylation status in glioblastoma brain tumours.glioblastoma brain tumours.

High resolution melt High resolution melt analysisanalysis

Perform a PCR using primers specific for Perform a PCR using primers specific for gene in questiongene in question

Following the PCR, heat the amplicon DNA Following the PCR, heat the amplicon DNA from around 50˚C up to around 95˚C. from around 50˚C up to around 95˚C.

At some point during this process, the At some point during this process, the melting temperature of the amplicon is melting temperature of the amplicon is reached and the two strands of DNA reached and the two strands of DNA separate or “melt” apartseparate or “melt” apart

The dsDNA is labelled with a fluorescent The dsDNA is labelled with a fluorescent dye, which fluoresces brightly. As the stands dye, which fluoresces brightly. As the stands separate the intensity of the dye is reduced separate the intensity of the dye is reduced and this reduction is measured in real time and this reduction is measured in real time producing a melt curveproducing a melt curve

High resolution melt High resolution melt analysis cont..analysis cont..

BGL is currently developing this technology BGL is currently developing this technology for testing of MPL and JAK2 Exon 12 for testing of MPL and JAK2 Exon 12 mutations in MPNs but many papers have mutations in MPNs but many papers have used it for BRCA1 and BRCA2 mutation used it for BRCA1 and BRCA2 mutation screening and TP53 screening in breast and screening and TP53 screening in breast and ovarian cancer which we are ?hoping to ovarian cancer which we are ?hoping to develop in the future.develop in the future.

http://en.wikipedia.org/wiki/High_Resolution_Melt

ReferencesReferences

Van Dongen et al., Design and Van Dongen et al., Design and standardisation of PCR primers and protocols standardisation of PCR primers and protocols for detection of clonal immunoglobulin and T for detection of clonal immunoglobulin and T cell receptor gene recombinations in suspect cell receptor gene recombinations in suspect lymphoproliferations. Leukemia, 2003. 17: lymphoproliferations. Leukemia, 2003. 17: 2257-23172257-2317

Liu H et al., A practical study for the routine Liu H et al., A practical study for the routine use of BIOMED-2 PCR assays for the use of BIOMED-2 PCR assays for the detection of B and T cell clonality in detection of B and T cell clonality in diagnostic haematopathology. Br J Haematol. diagnostic haematopathology. Br J Haematol. 2007. 138(1):31-432007. 138(1):31-43

AcknowledgementsAcknowledgements

Eileen Roberts (Head of Department, Eileen Roberts (Head of Department, BGL)BGL)

Dr Jerry Hancock (Molecular Oncology, Dr Jerry Hancock (Molecular Oncology, BGL)BGL)

Helen, Rich, Paul, Jess and Adelea Helen, Rich, Paul, Jess and Adelea (Molecular Oncology team)(Molecular Oncology team)

Dr Nicholas Rooney (Cellular Pathology)Dr Nicholas Rooney (Cellular Pathology) Konstantin Sidelnikov (InVivoScribe Konstantin Sidelnikov (InVivoScribe

Technologies, Inc) Technologies, Inc)