immunogenetics methodological considerationsimmunogenetics methodological considerations eric...

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Immunogenetics

Methodological considerations

ERIC international workshop

Biomarkers in CLL : the art of synthesis

Belgrade, March 16-17, 2018

Anton W. Langerak,

Laboratory Medical Immunology, Dept. Immunology

Erasmus MC, Rotterdam

[email protected]

Immunogenetics

Analysis of immunoglobulin (IG) / T-cell receptor (TR) rearrangements

exploiting the unique “molecular signatures” for different applications:

research

B/T cell ontogeny, immunodeficiency, autoimmune disease, hematological disease

clinical diagnostics

• clonality assessment

• MRD monitoring

• (clonal) repertoire analysis

Immunogenetics

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1 2 3 4 5 6 66 1 2 3 4 1 2 3 4 5 6

VH DH JH Cms

DJ joining

VD-J joining

precursor IGH mRNA

mature IGH mRNA

transcription

RNA splicing

V DJ Cm

27

IgH

IgL IgL

V V

C C

J J

IgHV V

D D

J J

C C

C

C

C

C

C

C

CD

79

a

CD

79

b translation

IG genes : a unique set of genes

5 4

- -

> 50 > 40

Ig Ig

> 1012Estimation of total primary receptor repertoire

++Junctional region

diversity

> 5 x 106

Combinatorial

diversity

6- J genes

27- D genes

> 100- V genes

Number of genes

IgH

Human IG repertoire

>> 1012Further diversification into SHM-shaped repertoire

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IG gene analysis in CLL

IGHV mutation analysis

optimization at two levels

• technical protocol to generate a reliable sequence

- avoid missing sequences

- avoid non-reliable sequence

• interpretation and conclusion to generate a reliable report

- correct clinical interpretation

IG analysis: from patient to sequence

Parameters

• Material

• Cell source

• Anticoagulant

• Work-up of cells

• Type of nucleic acid

• PCR methodology

• PCR protocol

• Taq polymerase

• PCR primers

• Processing and clonality

• Sequencing

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• peripheral blood

• bone marrow (idem)

• lymph node

• other

Survey prev. workshop (n =63)

0

10

20

30

40

50

60

PB BM LN other

Source of cells

other

frozen, paraffin, biopsies

Cell sourceParameters

• Material

• Cell source

• Anticoagulant



• PCR methodology

• PCR protocol

• Taq polymerase

• PCR primers


• Sequencing

0

5

10

15

20

25

30

35

40

45

50

EDTA CPT heparinized other

Anticoagulant

• EDTA tubes

• CPT tubes

• heparinized tubes

• other



AnticoagulantParameters

• Material

• Cell source

• Anticoagulant



• PCR methodology

• PCR protocol

• Taq polymerase

• PCR primers


• Sequencing

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other

FFPE, whole blood, red cell

lysis, osmotic analysis

0

5

10

15

20

25

30

35

40

45

Fi col l cel l susp other

W orkup o f cells

• Ficoll gradient (PB / BM)

• cell suspension of biopsy (e.g.

for flow analysis)

• other



Work-up of cellsParameters

• Material

• Cell source

• Anticoagulant



• PCR methodology

• PCR protocol

• Taq polymerase

• PCR primers


• Sequencing

0

5

10

15

20

25

30

gDNA only RNA / cDNA only both

Nucleic acid



Parameters

• Material

• Cell source

• Anticoagulant



• PCR methodology

• PCR protocol

• Taq polymerase

• PCR primers


• Sequencing

• genomic DNA

• RNA / complementary DNA

Type of nucleic acid Quantity of gDNA / RNA

-mostly 100-500 ng gDNA

(range 1 ng – 1 ug)

-mostly 1 ug RNA for cDNA

reaction (range 400 ng – 2 ug)

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advantage disadvantage

gDNA -more optimal for long-distance -unproductive

transport rearrangement can

-use of archival material also be amplified

RNA/cDNA -identifies mostly only -reverse transcription

productive rearrangement step required

-allows isotype identification

no scientific rationale for choosing gDNA or RNA/cDNA

(But advisable to use similar type of nucleic acid in multi-center trials)


Type of nucleic acid



Unproductive rearrangements are

not restricted to gDNA

RNA / cDNA: 1.6%

gDNA: 13.9%

Single unproductive

RNA / cDNA: 0.4%

gDNA: 0.8%

Langerak, Leukemia 2011

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Double rearrangements are not

restricted to gDNA

RNA / cDNA: 3.8%

gDNA: 14.8%

RNA / cDNA: ~1/3

gDNA: ~2/3

Langerak, Leukemia 2011

• no need for Taq enzyme with proofreading capacity

• number of independent reactions 1 or 2

(Taq polymerase errors are very rare)

• existing protocols optimized for applied PCR primer sets

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5

10

15

20

25

multiplex single both

PCR strategy


Parameters

• Material

• Cell source

• Anticoagulant



• PCR methodology

• PCR protocol

• Taq polymerase

• PCR primers


• Sequencing


PCR protocol and Taq polymerase

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L exon – VDJ exon – C exon

LVDJC transcript

Figure from Giudicelli and Lefranc, Ig gene analysis in CLL


Parameters

• Material

• Cell source

• Anticoagulant



• PCR methodology

• PCR protocol

• Taq polymerase

• PCR primers


• Sequencing

• IGHV leader primers in 2017 ERIC recommendation

LH (family-specific) – CH (Saohota, Blood 1996)

LH & VH (family-specific) – CH (Fais, J Clin Invest 1998)

• IGHV FR1 primers only in rare circumstances

FR1 consensus – JH (Aubin, Leukemia 1995)

FR1 multiplex – JH (BIOMED-2) (Van Dongen, Leukemia 2003)

• IGHV FR2 / FR3 primers : too short IGHV sequences NOT ACCEPTABLE

(only recommended upon negative leader / FR1 results, due to SHM)


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advantage disadvantage

HD analysis / -unlabeled products -lower detection limit

PAGE allow direct sequencing

GS analysis -higher detection limit -labeled products less

-optimal visualization optimal in sequencing

Clonality testing strategies – preferred methods

• heteroduplex analysis

• high resolution PAGE

• Gene Scan analysis / fragment analysis / spectratyping

• agarose electrophoresis : too low resolution DISCOURAGED

Parameters

• Material

• Cell source

• Anticoagulant



• PCR methodology

• PCR protocol

• Taq polymerase

• PCR primers


• Sequencing


Cases A, C, E, F, G

monoallelic

direct sequencing

Case D

bi-allelic

What is the next step ?

• direct sequencing

• single PCR sequencing

• gel excision sequencing

• cloning sequencing


HD interpretation

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Case B

What are possible scenarios ?

a. Small clone below detection limit

follow-up sample

b. Clone is present, but not

recognized different primer set

required

c. Lymphocytosis not due to CLL

clone check immunophenotype


HD interpretation

Direct sequencing

• one product from multiplex PCR

starting from IGHJ / IGHC, then reverse sequencing via IGHV family primer

• one product from single PCR

sequencing via specific primers from both sides

Sequencing after gel excision + elution

• bi-allelic rearrangement : physical separation of products

Sequencing after subcloning

• final option, e.g. physical separation of biallelic rearrangements impossible

0

5

10

15

20

25

30

35

40

direct cloning gel excision

Sequencing strategy


Sequencing strategies


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0

10

20

30

40

50

60

same nested M13 other

Primers used for sequencing

0

10

20

30

40

50

forw ard reverse both

Strands sequenced

Parameters

• Material

• Cell source

• Anticoagulant



• PCR methodology

• PCR protocol

• Taq polymerase

• PCR primers


• Sequencing



IG gene analysis

Most relevant parameters

• Type of nucleic acid RNA / cDNA and/or gDNA

• PCR primers Leader primers (FR1 only in exceptional cases)

• Clonality analysis PAGE / HD analysis or GeneScan analysis

• Sequencing mostly direct (w or w/o gel excision)

reliable sequence from two strands

Many parameters: no clear scientific rationale for one or the other option

Some strategies show complementary value

Clonality testing is an essential phase in the strategy !

ERIC recommendations

Rosenquist, Leukemia 2017

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IG gene analysis


ERIC recommendation on report format

IG gene analysis


ERIC recommendation on report format

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Guidelines on IG gene analysis

IG gene analysis - certification

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IG gene analysis – support service

Acknowledgements

Uppsala Universitet, Uppsala

Richard Rosenquist

Lesley-Ann Sutton

Pitié-Salpétrière, Paris

Fréderic Davi

San Raffaele, Milan

Paolo Ghia

Nikea, Athens/ Piraeus

Chrysoula Belessi

G. Papanicolaou, Thessaloniki

Kostas Stamatopoulos

Anastasia Hadzidimitriou

CEITEC, Brno // University Kiel

Nikos Darzentas

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Kinderdijk, NL

Erasmus MC, Rotterdam

Ruud Meijers Ellen van Gastel Michele van der Klift

Alice Muggen Ingrid Wolvers Irene Groen-van Mourik

Martine Kallemeijn Joyce Schilperoord Tamara Wabeke

Fatemeh Saberi Hosnijeh Kim Heezen

Jorn Assmann Tamara Pesic

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