Workshop on immunoglobulin gene analysis in chronic

lymphocytic leukemia

Bethesda, USA, March 29th 2019

Reporting IG gene sequence data in clinical

diagnostics

Lesley Ann Sutton

Clinical genetics, Molecular medicine & surgery (MMK),

Karolinska Institutet, Stockholm, Sweden

Lesley-ann.sutton@ki.se

Reporting IGHV gene somatic hypermutation in CLL

Basic details Recommendations

Patient data Name Gender

Date of birth ID number

Tissue type/Molecule type Peripheral blood

Bone marrow

Sample arrival date -

Requesting physician -

Reporting IGHV gene somatic hypermutation in CLL

Parameter Recommendations

- Methodology

- Gene identification

- Productive rearrangement

- IGHV gene: % nucleotide identity to germline

- Subset identification

Reporting IGHV gene somatic hypermutation in CLL

Methodology

• gDNA or cDNA

• PCR primers - Leader, Biomed (FR1)

• PCR product analysis/clonality assessment

- PAGE, GeneScan, Heteroduplex

• Sequencing strategy - direct Sanger, subcloning, NGS

• Bioinformatic tools - IMGT, Arrest/AssignSubsets

Technical details: an essential requirement for

all clinical genetic tests

Reporting IGHV gene somatic hypermutation in CLL

Usable sequences = productive rearrangements

They do not involve pseudogenes

They do not carry stop codons

They are in-frame

SHM IG gene analysis: only prognostic for productive rearrangements

Unproductive rearrangements can be included on the report: mention the reason for being unproductive (OF, stop codon etc.)

Reporting IGHV gene somatic hypermutation in CLL

Gene identification - parameters to consider

Genes – State the IGHV, IGHD and IGHJ gene and allele

VH CDR3 – check that the IG VDJ junction is in-frame with no stop codons

Check if the VH CDR3 anchors are present (C-104 & W-118)

SHM - Percentage of identity of the IGHV gene and allelic

ratio of aligned nucleotides

Stereotypy - Check if the rearranged sequence belongs to a stereotyped subset (#1, #2, #4 or #8)

Reporting IGHV gene somatic hypermutation in CLL

Gene identification - parameters to consider

Reporting IGHV gene somatic hypermutation in CLL

IGHD gene assignment is not always possible

Reporting IGHV gene somatic hypermutation in CLL

Percentage identity to the germline

• The % identity should always be reported, not only mutated/unmutated. Classification:

M-CLL < 98%; U-CLL ≥ 98%

• Borderline when IG gene mutational status is between 97-97,9%

• Counted from IMGT codon 1 to 104

• Automatically given by IMGT/V‐QUEST.

Reporting IGHV gene somatic hypermutation in CLL

BCR stereotypy - first step IMGT

*Subset #1 - IGHV1/5/7 - 13 AA VH CDR3

*Subset #2 – IGHV3-21 - 9 AA VH CDR3

*Subset #4 - IGHV4-34 - 20 AA VH CDR3

*Subset #8 – IGHV4-39 – 18 AA VH CDR3

Reporting IGHV gene somatic hypermutation in CLL

BCR stereotypy - second step Arrest/AssignSubset

http://www.ericll.org/guidance-toolsig/

http://www.ericll.org/wp-content/uploads/2017/10/Technical-Report-Guidelines.pdf

ERIC Guidance tools

Reporting IGHV gene somatic hypermutation in CLL

Cases difficult to categorize

Reporting IGHV gene somatic hypermutation in CLL

• The vast majority of cases will be categorized into M-CLL or U-CLL

But!!! A small fraction will be difficult to categorize

Cases difficult to categorize – Multiple rearrangements

Reporting IGHV gene somatic hypermutation in CLL

• 2% of 4154 cases carried double productive rearrangements (Langerak et al,

Leukemia 2011).

• More frequent when using gDNA.

• Usually only one rearrangement transcribed, however:

– Allelic exclusion or ’allelic inclusion’ has been described.

– Biclonality may occur rarely.

Cases difficult to categorize – Multiple rearrangements

Reporting IGHV gene somatic hypermutation in CLL

Double rearrangements: 435/4154 10.5%

•cDNA 61/1628

•gDNA 374/2526

Productive + unproductive: 350/435 80%

Double productive: 85/435 20%

Langerak et al, Leukemia 2011

Cases difficult to categorize – Multiple rearrangements

Reporting IGHV gene somatic hypermutation in CLL

Langerak et al, Leukemia 2011

Productive + unproductive 350/435 80%

Similar mutational status 326/350 93%

UM productive + M unproductive 17/350 5%

M productive + UM unproductive 7/350 2%

Cases difficult to categorize – Multiple rearrangements

Reporting IGHV gene somatic hypermutation in CLL

Langerak et al, Leukemia 2011

•

Double productive 85/435 20%

•Similar mutational status 56/85 66%

Discordant mutational status 29/85 34% (0.7% of total)

•

• Same mutation status

- Both productive

- one productive and one unproductive

Cases difficult to categorize – Multiple rearrangements

Reporting IGHV gene somatic hypermutation in CLL

Clinical interpretation is straightforward!

•

Cases difficult to categorize – Multiple rearrangements

Reporting IGHV gene somatic hypermutation in CLL

Divergent mutation status

Productive mutated IGH rearrangement & unproductive

unmutated IGH rearrangement

Interpreted and reported as mutated

•

Cases difficult to categorize – Multiple rearrangements

Reporting IGHV gene somatic hypermutation in CLL

Divergent mutation status

Productive unmutated IGH rearrangement & productive mutated

IGH rearrangement

Interpretation unknown

Due to the amplification of two productive rearrangements which exhibit discordant IGHV

mutational status, it is not possible to give a prognostically relevant interpretation

•

Cases difficult to categorize – Multiple rearrangements

Reporting IGHV gene somatic hypermutation in CLL

Divergent mutation status

Productive unmutated IGH rearrangement & unproductive

mutated IGH rearrangement

Interpretation unknown

•

Cases difficult to categorize – Single unproductive

rearrangements

Reporting IGHV gene somatic hypermutation in CLL

gDNA and cDNA:

1. Repeat the analysis.

2. Use an alternative set of primers.

3. If gDNA has been used, amplify instead using cDNA.

4. Repeat with a new sample.

In most cases a productive rearrangment should be possible to amplify

If still only one unproductive rearrangement detected - no clinical association can be made

http://www.ericll.org/guidance-toolsig/

http://www.ericll.org/wp-content/uploads/2017/10/Technical-Report-Guidelines.pdf

ERIC Guidance tools

Reporting IGHV gene somatic hypermutation in CLL

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