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
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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