1. No category

How to report IG sequence data in clinical
routine - cases difficult to categorize
100%
Cumulative Proportion Surviving
80%
60%
IGHV mutated
IGHV unmutated
40%
IGHV3-21
20%
0%
0
24
48
72
96
120
144
168
192
Time/ months
Richard Rosenquist
Uppsala, Sweden
Mains topics
• Key data to include in the IGHV mutation
status report.
• Categories of ’problematic’ cases when the
IGHV mutation status cannot be easily
reported.
The IGHV mutation status report
• Laboratories conducting IGHV mutation
analysis should report their findings in a
consistent way.
• This will facilitate comparison of data
between laboratories, especially in multicentre treatment trials.
Key data in the clinical report
Data to be included in the IGHV mutation status report in CLL
Category
Information to include
Basic data:
Patient data: name, date of birth and/or id number
Tissue type: peripheral blood or bone marrow
Sample arrival date
Referral doctor
Technique:
gDNA or cDNA
PCR primers (e.g. BIOMED2 primers)
Sequencing strategy: direct sequencing or, albeit rarely, subcloning
Results:
IGHV/IGHD/IGHJ gene usage
% sequence identity to germline
Reading frame – productive/unproductive rearrangement
Conclusion:
Should include interpretation of data (e.g. mutated/unmutated rearrangement or borderline) and
clinical association (e.g. good/poor risk or not known)
Basic data
• Patient data:
– name, date of birth and/or id number
• Tissue type:
– peripheral blood or bone marrow
• Sample arrival date
• Referral doctor
Technique
• gDNA or cDNA
– gDNA can give several rearrangements
• PCR primers
– BIOMED2 primers, leader primers
• Sequencing strategy:
– direct sequencing or, rarely, subcloning
Technical data is essential for all genetic tests!
Results I
• IGHV, IGHD, and IGHJ gene usage
– Summarized in the report by IMGT/V-QUEST.
– The best alignment should be reported.
– If two allelic variants give identical homology, note
down the first alternative
Results II
• Use the option in IMGT/V-QUEST to compare with all
eligible D genes. Highest score should be reported.
• If unmutated rearr. (100%) 2 instead 4 mutations allowed.
Results III
• In a proportion of cases the IGHD gene usage is not
possible to assign (e.g. stereotyped IGHV3-21 cases).
Results IV
• Gene usage data is important considering the finding that
IGHV gene usage/stereotypy may influence prognosis.
IGHV3-21
Ghia et al, Blood 2005
IGHV1/5/7
Stamatopoulos et al, Blood 2007
Results V
• Percentage identity
– The % identity should always be reported, not
only mutated/unmutated.
– Counted from IMGT codon 1 to 104
– Automatically given by IMGT/V-QUEST.
– May be additional nucleotides in IMGT codon
105-107 but potential mutations have a minor
effect (<0.5%).
Results VI
• ’Borderline’ mutation status
– CLL cases near the 98% cutoff
have varying clinical outcome
– 97%-99%.
– If FR1 primers have been
employed, the rearrangement
should be reanalyzed with
leader primers.
Hamblin et al, BJH 2007
Results VII
• Functionality
– Productive rearrangement
– Unproductive rearrangement
• Pseudogenes
• Out-of-frame rearrangement (deletions/insertions)
• Stop codons introduced by somatic hypermutation
– Multiple rearrangements
• Especially gDNA
Conclusion
• Mutated or unmutated rearrangement
• Cutoff used (e.g. 98%).
• Functionality should be mentioned, in
particular if two rearrangements.
• Clinical association?
– IGHV gene usage/stereotypy
– Borderline cases
Cases not possible to classify - state clearly that the
prognostic implication cannot be determined!
Example laboratory report for IGHV mutation status
Determination of immunoglobulin heavy variable (IGHV) gene
mutational status in chronic lymphocytic leukemia
Patient name: _________________________
Date of birth (DD-MM-YYYY): _____________
Arrival date (DD-MM-YY): _____________
Sample type: Peripheral blood
Referral doctor: R Brandell
Technique:
PCR amplification using framework 1 BIOMED2 primers and cDNA.
Direct sequencing.
Results:
IGHV:
IGHD:
IGHJ:
3-72
2-2
6
Identity:
95.7%
Functionality: Productive
Conclusion:
This CLL sample displays a productive IGHV mutated rearrangement. Mutated IGHV genes (<98%
identity) have been associated with good clinical outcome.
Dr Richard Rosenquist
Examples of conclusions I
A. Unmutated IGHV rearrangement
Results:
IGHV:
IGHD:
IGHJ:
1-69
3-3
4
Identity:
100%
Functionality: Productive
Conclusion:
This CLL sample displays a productive IGHV unmutated rearrangement. Unmutated IGHV
genes (≥98% identity) have been associated with poor clinical outcome.
B. Mutated IGHV3-21 rearrangement
Results:
IGHV:
IGHD:
IGHJ:
3-21
6
Identity:
96.7%
Functionality : Productive
Conclusion:
This CLL sample displays a productive IGHV3-21 mutated rearrangement (<98% homology)
with a stereotyped CDR3 (9 codons). The presence of a mutated IGHV3-21 with stereotypy
has been associated with poor clinical outcome.
Examples of conclusions II
C. Mutated ‘borderline’ rearrangement
Results:
IGHV:
IGHD:
IGHJ:
3-48
2-21
3
Identity:
97.8%
Functionality: Productive
Conclusion:
This CLL sample displays a productive, mutated IGHV rearrangement with borderline identity
(close to the 98% cut-off). Caution should be taken when interpreting the clinical correlation.
D. Two unmutated, productive IGHV rearrangements
Results:
Rearrangement 1:
IGHV:
1-69
IGHD:
3-16
IGHJ:
6
Rearrangement 2:
IGHV:
3-30
IGHD:
3-3
IGHJ:
4
Identity:
99.6%
Functionality: Productive
Identity:
100%
Functionality: Productive
Conclusion:
This CLL sample displays two unmutated productive IGHV rearrangements and would be
interpreted as expressing unmutated IGHV genes. Unmutated IGHV genes (≥98% identity)
have been associated with poor clinical outcome.
Examples of conclusions III
E. One productive and one unproductive, unmutated IGHV rearrangements
Results:
Rearrangement 1:
IGHV:
1-69
IGHD:
3-3
IGHJ:
6
Rearrangement 2:
IGHV:
4-34
IGHD:
3-22
IGHJ:
4
Identity:
99.6%
Functionality: Productive
Identity:
100%
Functionality: Unproductive
Conclusion :
This CLL sample displays a productive IGHV unmutated rearrangement and an unproductive
IGHV unmutated rearrangement. Altogether, the interpretation is a case with unmutated
IGHV genes. Unmutated IGHV genes (≥98% identity) have been associated with poor clinical
outcome.
F. Unproductive IGHV rearrangement
Results:
IGHV:
IGHD:
IGHJ:
1-3
3-3
6
Identity:
100%
Functionality: Unproductive
Conclusion:
This CLL sample displays only an unproductive IGHV unmutated rearrangement (≥98%
identity). At present, the clinical correlation for this case cannot be defined.
Cases difficult to categorize
• The vast majority will be categorized into
mutated or unmutated.
• A small fraction will be difficult to
categorize.
• Two main types:
– Single unproductive rearrangement
– Double rearrangements with divergent
mutation status.
Single unproductive rearrangements I
• IGHV pseudogenes
• Stop codons
Single unproductive rearrangements II
• Out of frame - deletions/insertions
• Absent CDR3 anchors - G-X-G motif intact?
Strategy - only single unproductive
rearrangement
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 rearrangement should
be possible to amplify.
If still only one unproductive rearrangement
– no clinical association can be made!
Multiple rearrangements
• 1.5% of cases carried double productive
rearrangements (Murray et al, Blood 2008).
• More frequent when using gDNA.
• Usually only one rearrangement
transcribed, however:
– Allelic exclusion or ’allelic inclusion’ has been
described.
– Biclonality may occur rarely.
Lack of allelic exclusion (allelic
inclusion)
• Often employed by autoreactive B cells.
Kenny JJ et al. Autoreactive B cells escape clonal deletion by expressing
multiple antigen receptors. J. Immunol. 2000.
• Frequent feature in the normal marginal-zone.
Li Y, et al. Autoreactive B cells in the marginal zone that express dual
receptors. J. Exp. Med. 2002.
• Previously reported in CLL, at least at transcript level.
Rassenti LZ, Kipps TJ. Lack of allelic exclusion in B cell chronic
lymphocytic leukemia. J. Exp. Med. 1997.
Biclonality
• Rare, but has been reported up to 5% in chronic
lymphoproliferative disorders.
• More frequent in atypical CLL. Were related to
increased WBC, splenomegaly and early treatment
(Sanchez, Blood 2003).
• Can only be formally proven if different light chain
expression.
Most cases with 2 rearrangements does not
represent biclonal cases!
Multiple rearrangements
• Same mutation status:
– Both productive
– One productive and one unproductive
• The clinical interpretation is
straightforward!
Divergent mutation status
• 14 of 36 CLL cases with double productive
rearrangements showed discordant
mutation status (out of ~2000 cases).
• How to interpret these cases???
Divergent mutation status
• Productive mutated IGH rearrangement +
unproductive unmutated IGH
rearrangement
– Will be interpreted as mutated.
• Productive unmutated IGH rearrangement +
unproductive mutated IGH rearrangement.
– Interpretation unknown.
Unmutated rearrangements with low level of
somatic hypermutation
Case
IGHV
%
Functionality
Swe-145-I
Swe-145-II
IGHV3-23
IGHV3-66
98.2
96.4
Productive
Unproductive
Unmutated
Mutated
Swe-267-I
Swe-267-II
IGHV3-48
IGHV1-69
98.6
96.4
Productive
Unproductive
Unmutated
Mutated
Clinical interpretation cannot be made in these cases!
The ERIC database
• More ’problematic’ cases has to be collected
to investigate the clinical association.
• The ERIC database has been created to
facilitate an internationell collaborative
effort.
• Dr Anton Langerak will tell us more!
FRANCE
Paris
Frédéric Davì
ITALY
Milan
Paolo Ghia
GREECE
Thessaloniki - Athens
Kostas Stamatopoulos
Chrysoula Belessi
Thank you for your attention!