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!
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