BIOMARKERS IN CHRONIC LYMPHOCYTIC LEUKEMIA: THE ART OF SYNTHESIS - - PowerPoint PPT Presentation

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INTERNATIONAL WORKSHOP BIOMARKERS IN CHRONIC LYMPHOCYTIC LEUKEMIA: THE ART OF SYNTHESIS Session IV. Immunogenetics Interactive case session Frederic Davi Professor | Hpital Piti - Salptrire Andreas Agathangelidis post-doc | Institute

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Belgrade | March 16-17, 2018

INTERNATIONAL WORKSHOP BIOMARKERS IN CHRONIC LYMPHOCYTIC LEUKEMIA: THE ART OF SYNTHESIS Session IV. Immunogenetics Interactive case session

Frederic Davi Professor | Hôpital Pitié-Salpêtrière Andreas Agathangelidis post-doc | Institute of Applied Biosciences (INAB)

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Background

Belgrade | March 16-17, 2018

V D J V D J D J rearranged V Germline D-J joining V-DJ joining

CDR3 SHM status

How?

germline V

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VH CDR3

tgtgcaaaag gacc gactacggtggtaactcc gggg tttgactactgg

C A K G P T T V V T P G F D Y W

tgt gca aaa gga ccg act acg gtg gta act ccg ggg ttt gac tac tgg

104 118 C W V N1 D N2 J

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SHM analysis – clinical relevance

Hamblin et al., Blood 1999 Survival was significantly worse for patients with unmutated VH genes irrespective of stage. Damle et al., Blood 1999 Patients in the unmutated group responded poorly to chemotherapy and had shorter survival.

98% cut-off

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CLL: not only M and UM cases?

Borderline cases:

not intermediate

prognosis

mix of cases with

aggressive and indolent disease

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An Introduction of IMGT/V-QUEST tool
get familiar with the sequence submission process
review basic parameters of the tool
Interactive exercises
analysis of IGH rearrangement sequences
sequence annotation and feature characterization
Sequence interpretation and results

Belgrade | March 16-17, 2018

Aim

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IMGT/V-QUEST Access: http://www.imgt.org/

Belgrade | March 16-17, 2018

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Sequence submission

Submit a single or a batch of sequences (up to 50) manual copy/paste or file upload In FASTA format : :

Belgrade | March 16-17, 2018

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Results retrieval

Information about the type of output files provided in each case: IMGT/V-QUEST Documentation http://www.imgt.org/IMGT_vquest/share/textes/imgtvquest.html

Belgrade | March 16-17, 2018

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>Sequence A1

caggtgcagctacagcagtggggcgcaggactgttgaagccttcggagaccctgtccctc acctgcgctgtctatggtgggtccttcagtggttactactggagctggatccgccagccccca gggaaggggctggagtggattggggaaatcaatcatagtggaagcaccaactacaaccc gtccctcaagagtcgagtcaccatatcagtagacacgtccaagaaccagttctccctaaag ctgagctctgtgaccgccgcggacacggctgtgtattactgtgcgagaggtctacctcttttgg agtggttattgggtccttactactactactacggtatggacgtctggggccaagggaccacgg tcaccgtctcct

Belgrade | March 16-17, 2018

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Sequence A1

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Sequence A1

Belgrade | March 16-17, 2018

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IGHV4-34*01 IGHD3-3*01 IGHJ6*02 identity 99,65% in-frame VH CDR3 length 22 aa

→ productive, unmutated IGH

Results

Belgrade | March 16-17, 2018

Prognosis?

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>Sequence A2

caggtgcagctggtgcagtctggagctgaagtgaagaagcctggggcctcagtgaaggtc tcctgcaaggcttctggttacacctttacgaattatggtatcggctgggtgcgacaggcccctg gacaagggcttgagtggatgggatggatcagcggttacaatggtgatacaaactatgcaca gaagttccaggacagagtcaccatgaccacagacacatccacgagcacagcctatatgg acctggggagcctgagatctgacgacacggccgtgtattactgtgcgagagtagtggctac gctcccccccgtctactggggccagggaaccctggtcaccgtctcctca

Belgrade | March 16-17, 2018

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Sequence A2

Belgrade | March 16-17, 2018

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Sequence A2

Belgrade | March 16-17, 2018

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IGHV1-18*01 IGHD5-12*01 IGHJ4*02 identity 95,14% in-frame VH CDR3 length 11 aa

→ productive, mutated IGH

Sequence A2

Belgrade | March 16-17, 2018

Prognosis?

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>Sequence A3 gaggtgcagctggtggagtctgggggaggcttggtccagcctggagggtccctgagactct cctgtgcagcctctggattcaccttcagtgaccactacatggactgggtccgccgggctcca gggaaggggctggagtgggttggccgtactagaaataaagctaacagttacaccacaga gtacgccgcgtctgtgaaaggcagattcaccatctcaagagatgattcaaacaactcactgt atctacaaatgagcagcctgaaaatcgaggacacggccgtgtattactgtgttcgagtctttat acctacctccacacccgactactggggccagggaaccctggtc

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Sequence A3

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IGHV3-72*01 IGHD3-16*02 IGHJ4*02 identity 97,62% in-frame VH CDR3 length 12 aa

→ productive, borderline mutated IGH

Sequence A3

Prognosis?

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Ghia P et al. ERIC recommendations on IGHV gene mutational status analysis in CLL. Leukemia 2007;21: 1-3

as for any mathematical cutoff value applied to a biological phenomenon, one has to be cautious when dealing with ‘borderline cases’

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>Sequence A4 gaggtgcagttggtggagtctgggggaggcctggtcaagcctggggggtccctgagac tctcctgtgcagcctctggattcaccttcagtagctatagcatgaactgggtccgccaggct ccagggaaggggctggagtgggtctcatccattactattagtagtggttacatatactacg cagactcagtgaggggccgattctccctctccagagacaacgccaagaactcactgtat ctgcaaatgaacagcctgcgagccgaagacacggctgtgtattactgtgcgagagatg ccaacggtatggacgtctgggggcaaggg

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Sequence A4

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Sequence A4

IGHV3-21
Mostly IGHJ6
VH CDR3 : 9 aa
D or E at position VH

CDR3-107 This rearrangement represents a CLL subset #2 case.

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IGHV3-21*01 IGHD3-16*02 IGHJ6*01 identity 96,88% in-frame VH CDR3 length 9 aa

→ productive, mutated IGH, subset #2

case

Sequence A4

Prognosis?

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>Sequence A5 gagatgcagctggtggagtctgggggaggcctggtcaaccctggggagtccctgagactct cctgtgcatcctctggattcaccctcagtagctatagtatgagctgggtccgccaggctccag ggaaggggctggagtgggtctcatcccttagtagtagtagtggttacgtatactacgcagact cagtgaagggccgattcaccatctccagagacaacgccatgaactcactgtatctgcaaat gaacagcctgagagccgaggacacggctgtgtattactgtgcgagagatgccaacggtat ggacgtctgggggcaagga

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IGHV3-21*01 IGHD2-2*01 IGHJ6*02 identity 96,18% in-frame

→ productive, mutated IGH

Sequence A5

Belgrade | March 16-17, 2018

Prognosis?

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Patients belonging to subset #2 have poor prognosis irrespective of SHM status and this should be stated clearly in the report to the clinician.

Sequence A5

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>Sequence B2

gaggtgcacctggtggagtctgggggaggcctggtcaacccgggggggtccctcagactc tcctgtgcagcctctggattcaccttcagtaattttagcatgacctgggtccgccaggctccag ggcaggggctggagtgggtctcatccatggagtgggtctcatccattagtagtagtagtaatt acatatactacgcagactcagtgaagggccgattcaccatctccagagacaacgccaag aactcactgtatctgcaattgaacagcctgagaggcgaggacacggctgtttattactgtgcg agaatggtggcttccaagtacccaccatactactttgacttctggggcccgggaaccctggtc accgtctcctcag

Belgrade | March 16-17, 2018

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Sequence B2

Belgrade | March 16-17, 2018

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IMGT/V-QUEST provides warnings to alert the users, for the possibility that potential insertions or deletions are present in the sequence

Sequence B1

http://www.imgt.org/IMGT_vquest/share/textes/IMGTvquest-warnings.pdf

Warning Objective V‐GENE and allele: low V‐REGION identity percentage with the closest V germline less than 85% of identity with the closest germline V‐ GENE and allele: this may indicate that the V‐GENE and allele name could be not reliable. V‐GENE and allele: 2nd‐CYS 104 not identified the sequence junction may not de reliable. V‐GENE and allele: different CDR1‐IMGT and/or CDR2‐IMGT amino acid lengths compared to closest V germline this may indicate that the V gene and allele name could be not reliable. Belgrade | March 16-17, 2018

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Sequence B1

Back to the submission form
Enable the option at the “advanced parameters” tab
Note that now you can simultaneously analyze a batch of max. 10

sequences

Belgrade | March 16-17, 2018

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Sequence B2

Belgrade | March 16-17, 2018

18nt insertion detected in VH FR2 region

not causing frameshift  Potentially productive IGH rearranged sequence % identity to germline is calculated considering the insertion as one mutational event

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Sequence B2 IGHV3-21*01 IGHD5-12*01 IGHJ4*02 identity 95.14% in-frame VH CDR3 length 15

→ productive, mutated IGH rearrangement

Belgrade | March 16-17, 2018