IG Cer'fica'on. Andreas Agathangelidis Ins'tute of Applied Biosciences, CERTH ADFDHHHHHHHHHHHHHHHH

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1 IG Cer'fica'on Andreas Agathangelidis Ins'tute of Applied Biosciences, CERTH ADFDHHHHHHHHHHHHHHHH

2 Round 3 Project lead by: CERTH/INAB Deadline to apply: 15 September 2017 Sample shipment: 5 December 2017 Ongoing

3 Background Phase 1 - Registering current prac'ces through an online survey of interested labs. Phase 2 - Laboratory and bioinforma'cs analysis: (i) transfer online of a research planorm, i.e. tools, documentakon and guidelines; Q&A forum; report templates and system to receive them; (ii) prepara'on of quality control material - genomic DNA from 5 CLL cases of diverse mutakonal status; (iii) shipment of quality control material to all parkcipakng labs who have answered the survey; (iv) analysis in evaluated lab and sending of report template to ERIC Reference lab within 3 months. Phase 3 - Evalua'on & decision Assessment of the results by the IgCLL group in terms of both the analysis per se (e.g. examinakon of sequencing runs) and IG sequence interpretakon. If results are OK: provide ERIC Cer'fica'on; If results are not OK: provide guidance and offer second chance.

4 ROUND 2 Project details & deadlines: Project lead by: CERTH/INAB (Greece) Original Applicants: 35 Par'cipa'ng centers: 33 Number of par'cipa'ng countries: 17 Samples Sent to Par'cipants: 15 March 2017 Deadline to submit results: 15 May 2017

5 33 par'cipa'ng centres 17 par'cipa'ng countries 8 Par'cipa'ng countries ADFDHHHHHHHHHHHHHHHH

6 the QC sequences - overview Sample ID IGHD gene IGHJ gene % iden'ty to germline SHM status Comments ERIC2-1 V3-23*01 or V3-23D*01 D3-3*01 J4* borderline ERIC2-2 V1-69*01 or V1-69D*01 D3-3*01 J6* unmutated ERIC2-3 V3-23*01 or V3-23D*01 D6-13*01 J4* mutated ERIC2-4 V3-53*01 D5-24*01 J4* mutated ERIC2-5 V3-21*01 D6-19*01 J6* borderline in- frame delekon of 3 nucleokdes in VH CDR2 1. in- frame inserkon of 12 nucleokdes in VH FR3 2. stereotyped subset #2

7 the QC sequences points to consider Sample ID IGHD gene IGHJ gene % iden'ty to germline SHM status Comments ERIC2-1 V3-23*01 or V3-23D*01 D3-3*01 J4* borderline Following the 98% germline idenkty cut- off value which is used for discriminakng CLL cases into the IG- mutated or IG- unmutated category, case ERIC2-1 belongs to the IG- mutated category. However, the idenkty percentage is close to the cut- off and, thus, the case can be considered as borderline- mutated. In such cases, caukon is warranted regarding the precise prognoskc implicakons.

8 the QC sequences points to consider Sample ID IGHD gene IGHJ gene % iden'ty to germline SHM status Comments ERIC2-2 V1-69*01 or V1-69D*01 D3-3*01 J6* unmutated An unproduckve IGHV3-13*05/IGHD5-12/IGHJ3*02 with an out- of- frame junckon and stop codons within the VH CDR3 and 100% idenkty to the germline IGHV3-15*05 gene could also be amplified along with the produckve rearrangement. Since the idenkty of both rearrangements was idenkcal, the case is classified as IG- unmutated. Langerak et al. Leukemia 2011 Rosenquist et al. Leukemia 2017

9 the QC sequences points to consider Sample ID IGHD gene IGHJ gene % iden'ty to germline SHM status Comments ERIC2-3 V3-23*01 or D6-13*01 J4* mutated V3-23D*01 ERIC2-5 V3-21*01 D6-19*01 J6* borderline in- frame delekon of 3 nucleokdes in VH CDR2 1. in- frame inserkon of 12 nucleokdes in VH FR3 2. stereotyped subset #2 In cases with inser-ons and dele-ons, analysis on IMGT/V- QUEST gives a warning Difference of CDR1- IMGT and/or CDR2- IMGT amino acid lengths with the closest germline V- GENE and allele, hence promp-ng to perform a second analysis of the sequence with the selec-on of the op-on "Search for inser-ons and dele-ons in V- REGION" in "Advanced parameters" of the IMGT/V- QUEST Search page.

10 the QC sequences points to consider Sample ID IGHD gene IGHJ gene % iden'ty to germline SHM status Comments ERIC2-3 V3-23*01 or D6-13*01 J4* mutated V3-23D*01 ERIC2-5 V3-21*01 D6-19*01 J6* borderline in- frame delekon of 3 nucleokdes in VH CDR2 1. in- frame inserkon of 12 nucleokdes in VH FR3 2. stereotyped subset #2 This will lead to correct calcula-on of the germline iden-ty of the rearranged, where you have to keep in mind that the inser-on or dele-on will count as one one addi-onal muta-onal event [please see the IMGT/V- QUEST field V- REGION iden-ty % (with ins/del events) ].

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12 Only the use of leader primers allows for the amplificakon of the enkre sequence of the rearranged, thus enabling the true and complete level of SHM to be determined, and is the recommended choice. The use of primers annealing to the VH FR1 or even more downstream parts of the VH domain (for example, the VH FR2 or VH FR3) is not acceptable for determining the SHM status. That said, in rare cases when leader primers are unsuccessful at providing a product that can be sequenced and VH FR1 primers are used (not generally recommended), the report should indicate that the use of VH FR1 primers might undereskmate the total number of IGHV SHMs.

13 Accredita'on requirements Par'cipa'ng in ERIC IG survey Covering at least VH CDR1- VH CDR3 of all samples Covering VH FR1 VH CDR3 of all samples Iden'fica'on of sample ERIC2-1 as IGHV- mutated Raise cau'on about the borderline status of ERIC2-1 Iden'fica'on of sample ERIC2-2 as IGHV- unmutated Iden'fica'on of sample ERIC2-3 as IGHV- mutated Iden'fica'on of sample ERIC2-4 as IGHV- mutated Iden'fica'on of sample ERIC2-5 as belonging to stereotyped subset #2 Correct annota'on Provided Lab specific report Correct interpreta'on Finished and submibed analysis before the deadline Good laboratory prac'ce requirements

14 Overall assessment - Round 2 Passed: 29* Failed: 4 Total: 33 *for some labs, pending resolu-on and/or clarifica-on of certain issues

15 Current situa'on: 60 InsKtuKons 25 countries

16 Thank you for your 'me! For further informakon: hbp://