NGS IMMUNOGENETICS IN CLL RESEARCH. Andreas Agathangelidis Post-doc researcher Institute of Applied Biosciences, CERTH

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1 NGS IMMUNOGENETICS IN CLL RESEARCH Andreas Agathangelidis Post-doc researcher Institute of Applied Biosciences, CERTH ERIC & Euroclonality-NGS workshop Rotterdam, 24 November 2017

2 1. NGS Immunoprofiling of the T Cell Repertoire in CLL CLL: Bi-directional interactions between clonal cells and T cells Provision of important trophic signals for the survival and proliferation Accumulation of phenotypic and functional deviations Changes in the gene expression profile Reduced ability to boost effective cytotoxic responses against the leukemic clone Development of novel therapeutic protocols with immunomodulating agents that act at the level of the immune synapse

3 Study group 36 CLL cases 16 U-CLL, 20 M-CLL - subset #1 - subset #2 - subset #4 - mutated Igs non stereotyped - unmutated Igs non stereotyped before treatment & without symptoms of infection at sample collection 3 healthy donors

4 why focus on stereotyped subsets? subsets #1, #2 and #4 are: (i) homogeneous, (ii) distinct clinical entities

5 the TRBV gene repertoire in CLL is skewed TRBV12 3/12 4 TRBV29 1 TRBV19 TRBV5 1 TRBV6 5 5/53 TRBV genes = 33.3% of all repertoire other Identical most prevalent genes NGS vs Sanger

6 pronounced T cell repertoire oligoclonality healthy M-CLL U-CLL Subset 2 Subset 16 Subset 1 Subset

7 Overtime Analysis case P1 subset #1 PMNCs TRBV10-3 TRBV27 TRBV TRBV19 TRBV29-1 TRBV6-5 TRBV5-1 P1 - Timepoint 1 year 1 P1 - Timepoint 2 year 3 consistent TRBV gene frequencies

8 Overtime Analysis: persisting clonotypes case P1 subset #1 PMNCs 100% case P2 subset #4 t1: PMNCs, t2: CD4+, CD8+ % Clonotype frequency 75% 50% 25% year 1 P1 - Timepoint 2 - CD4 year 3 0% P1 - Timepoint 1 P1 - Timepoint 2 P1 - Timepoint 1 - PMNCs year 1 year 3 P1 - Timepoint 2 - CD8 persistent antigenic stimulation?

9 Common major clonotypes in subset #4 100% 100% 100% 100% 75% 75% 75% 75% 50% 50% 50% 50% 25% 25% 25% 25% 0% P1 #4 P2 #4 0% P3 #4 P4 #4 0% P7 #4 P8 #4 0% P5 #4 P6 #4 subset #4: IGHV4-34/IGKV2-30

10 Conclusions: Selection By Shared Antigenic Elements Pronounced oligoclonality of T cells Shared clonotypes Evidence of persisting antigenic selection Nature of the selecting antigens? Vardi et al. Leukemia 2017

11 2. Impact of BcR signaling inhibitors versus immunotherapy on the T cell compartment Scientific aim Study of the T cell receptor repertoire based on: (i) Therapeutic protocol (ii) Response to treatment Study group 16 patients with CLL: sampling before and 3 months after therapy Therapeutic protocol Patient no. Response to treatment +3 months FCR 5 CR Ibrutinib (ΙΒ) 9 PR Rituximab-Idelalisib (R-ID) 7 PR

12 T cell receptor repertoire FCR TRBV7-2 TRBV19 TRBV12-3/ TRBV29-1 TRBV6-5 IBRUTINIB TRBV7-2 TRBV19 TRBV12-3/ TRBV29-1 TRBV6-5 TRBV5-1 TRBV27 TRBV28 R-IDELALISIB TRBV5-1 TRBV27 TRBV28 TRBV20-1 TRBV10-3 TRBV TRBV29-1 TRBV5-1 TRBV27 TRBV19

13 Clonality before and after treatment Median frequency of 10 dominant clonotypes per sample % * FCR Ibrutinib R-Idelalisib * *p<0.05

14 Clonal evolution - Response to therapy IBRUTINIB R-IDELALISIB 100% 90% 80% 70% 60% 50% 40% 30% 100% 90% 80% 70% 60% 50% 40% 30% 20% 20% 10% 10% 0% before προ Ibrutinib µετά after Ibrutinib PR after µετά Ibrutinib CR 0% before προ R- R- Idelalisib µετά after R- µετά after R- Idelalisib Idelalisib PR CR

15 Repertoire changes FCR Ibru4nib R idelalisib P5 P4 P3 P2 P1 0% 20% 40% 60% 80% 100% P18 P17 P16 P15 P14 P13 P12 P11 P10 0% 20% 40% 60% 80% 100% P25 P24 P23 P22 P21 P20 P19 0% 20% 40% 60% 80% 100%

16 Conclusions T cell expansions are evident before and after treatment BcR signaling inhibitors and immunotherapy have different effects on the T cell compartment Immunotherapy leads to the renewal of the T cell repertoire and an increase of T cell clonality BcR signaling inhibitors do not affect the T cell clones The additional expansion of T cell clones on the cases with better response to treatment could mean that these clones anti-leukemic, at least in the case of R-ID Vardi et al. ASH 2017

17 Monoclonal B cell lymphocytosis (MBL) Small clonal expansions of B cells with the characteristic CLL cell phenotype MBL categories clinical/high-count MBL (HC-MBL) (0.5-5x10 9 clonal cells/l blood) evolves to CLL requiring treatment at a rate of 1-2% /year. general population/low-count MBL (LC-MBL) (<0.5x10 9 clonal cells/l blood) seems to remain stable over time. The relationship between LC-MBL and CLL is still obscure.

18 MBL CLL relationship Genetics Same cytogenetic aberrations with CLL (similar frequencies) Common driver somatic mutations in genes implicated in CLL pathogenesis (namely NOTCH1, SF3B1 etc.) Microenvironment LC-MBL differs substantially at the BcR repertoire level from HC-MBL and CLL that exhibit similar immunogenetic features Important similarities between HC-MBL and CLL characterized by biases in the T cell receptor repertoire

19 3. T cell receptor immunoprofiling of LC-MBL Scientific aim Assess the T cell landscape of LC-MBL and identify possible similarities with CLL Study group 50 cases of LC-MBL in total 2 sample groups: I. CLL-like LC-MBL (43 samples) II. Non CLL-like LC-MBL (7 samples)

20 T cell receptor repertoire analysis CLL-like LC-MBL TRBV29-1 Non CLL-like LC-MBL TRBV29-1 TRBV6-5 TRBV6-5 5/50 TRBV genes = 33,4% of the total repertoire TRBV19 TRBV12-3/ TRBV12-4 TRBV5-1 άλλο other 5/50 TRBV genes= 34% of the total repertoire TRBV19 TRBV12-3/ TRBV12-4 TRBV28 άλλο other CLL TRBV12-3/1 2-4 TRBV29-1 5/53 TRBV genes = ~30% of the total repertoire TRBV19 TRBV5-1 TRBV6-5 other άλλο other

21 TRBV repertoire comparisons Average frequency in CLL-like LC-MBL Average frequency in non CLL-like LC-MBL Average frequency in CLL Μέση συχνότητα εµφάνισης στη LC-MBΛ τύπου ΧΛΛ Μέση συχνότητα εµφάνισης στη LC-MBΛ άλλου τύπου Μέση συχνότητα εµφάνισης στη ΧΛΛ

22 T cell receptor clonality in LC-MBL Health donors 5,2% CLL 26,3% Non CLL-like LC-MBL 20,3% CLL-like LC-MBL 21,3% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

23 Common clonotypes 12 clonotypes between CLL-like LC-MBL samples 4 clonotypes between CLL-like/non CLL-like LC-MBL samples 1 clonotype between CLL-like LC-MBL/CLL 2 clonotypes between CLL-like LC-MBL/non CLL-like LC-MBL/CLL

24 Conclusions Immunogenetic characteristics of LC-MBL Biases in the T cell receptor repertoire Dominance of the same TRBV genes Similar degree of oligoclonality Very few common clonotypes between LC-MBL samples and even fewer between LC-MBL and CLL samples

25 Conclusions An overall similar to CLL immunogenetic scenario in LC-MBL: Interactions between T cells and antigens shapes the T cell receptor repertoire The nature of these antigens and their signals are probably different from those observed in CLL Agathangelidis et al. ASH 2017

26 4. Search for CLL-specific BcR stereotypes in LC-MBL Scientific aim Understand the role of the microenvironment in CLL ontogenesis identify CLL-specific IG sequences Study group 45 samples in total 2 main donor groups: I. LC-MBL donors Sorted MBL B cells from MBL donors PBMCs from MBL donors Sorted normal B cells from MBL donors II. Healthy/ LC-MBL negative 6 healthy donors from which: Sorted naïve B cells Sorted memory B cells Unsorted B cells

27 Higher degree of clonality in LC-MBL Sorted LC-MBL Average/ samples LC-MBL PBMCs Average/ samples Sorted B cells from LC-MBL Average/ samples Sorted naice B cells Average/ samples Sorted memory B cells Average/ samples Total B cells Average/ samples Total raw reads , Filtered-In reads ,5 Expanded clonotypes Singletons (1read) ,

28 B cell receptor clonality Total B cells 1,3% (0,8-1,8) Sorted memory B cells 4,8% (1,4-11) Sorted naïve B cells 1,5% (0,5-1,9) Sorted B cells from LC-MBL 2% (0-2,8) PBMCs from LC-MBL 14% (6-42) Sorted LC-MBL cells 77% (17-98) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

29 Common clonotypes (frequency >1%) 1 clonotype between sorted LC-MBL samples 1 clonotype between sorted LC-MBL and sorted B cells from LC-MBL samples 1 clonotype between sorted LC-MBL and PBMC from LC- MBL samples

30 Presence of stereotyped sequences among sample categories Sample category Sample number with stereotyped sequences Frequency Sorted LC-MBL 5/12 41% PBMCs from LC-ΜΒL 3/11 27% Sorted B cells from LC-MBL 7/9 78% Sorted naïve B cells 6/6 100% Sorted memory B cells 2/5 40% Total B cells 2/2 100%

31 Distribution of stereotyped sequences per sample category Stereotyped subset Sorted LC-MBL cells 12 samples PBMCs from 11 LC-MBL samples Normal B cells from 9 LC-MBL samples Sorted naïve B cells 6 samples Sorted memory B cells 5 Total B samples cells # # # # # # # 7C # # # # # 28A # # # 64B # # # # samples

32 Conclusions Clonality Greater degree of clonality in LC-MBL compared to samples from healthy donors Heterogeneity Healthy samples were polyclonal CLL-stereotyped sequences Very low frequency in a portion of samples Absence of strong correlations between sample categories and individual subsets (exception: #4) LC-MBL: more frequent in normal B cell samples that clonal B cell samples Agathangelidis et al. ASH 2017

33 Chrysi Galigalidou Elisabeth Vlachonicola Katerina Gemenetzi Evangelia Stalika Fotis Psomopoulos Anna Vardi Kostas Stamatopoulos Anastasia Hadzidimitriou Lydia Scarfò Alessandra Rovida Pamela Ranghetti Paolo Ghia Fred Davi Ton Langerak Fred Davi