mia Nicholas Chiorazzi The Feinstein Ins3tute for Medical Research - PowerPoint PPT Presentation

A Some mewhat Diffferent View of the the Gene ne3c 3c Portr rtrait ait of Chronic Lymp mphocy3c Leukemi mia Nicholas Chiorazzi The Feinstein Ins3tute for Medical Research Northwell Health Manhasset, NY

Acknowledgments UNIVERSITÀ Davide Bagnara DEGLI STUDI DI GENOVA Stefano Vergani Andrea Mazzarello Gerardo Ferrer Anthony Liew

Te Tenets § In general, the human genome is evolu2onarily designed to correct soma2c muta2ons that might occur § Cancer is a disease in which these protec2ve mechanisms ul2mately fail, resul2ng in gene2c derangements of specific, biologically important genes § CLL is the same in some ways and different in others. § B lymphocytes, the cells that become leukemic in CLL, need to deal with a different evolu2onarily design, i.e., the desired accumula2on of muta2ons in the genes coding for the an2gen-binding regions of an IG molecule by ac2va2on- induced deaminase (AID)

IG V-region gene mu muta3ons in CLL § Soma2c muta2ons of IGHV genes divide CLL clones/ pa2ents into two categories: U-CLL and M-CLL § U-CLL and M-CLL pa2ents differ in their clinical courses § IGHV muta2on status remains an important and reliable indicator of clinical course and survival in CLL

§ Almost invariably “IGHV muta2on status” is fixed. § Very rarely does a clone “switch” from U-CLL to M-CLL, although this can occur § However, Sanger sequencing studies indicate that the IGHV-D-J rearrangement of CLL clones can undergo intraclonal heterogeneity, even U-CLL cells ( Gurrieri et al. 2002; Bagnara et al. 2006; Volkheimer et al. 2007; SuSon et al. 2015) § AID, the gene responsible for IGHV-D-J muta2ons, is func2onally ac2ve in CLL. § If inten2onally upregulated, this enzyme will mutate CLL V regions, even in U-CLL cells.

Ques3ons: § How extensive is the ongoing IGHV-D-J muta2on process in CLL? § Can this ongoing process be used to predict the likelihood that muta2ons, at least those caused by AID, will occur at sites throughout the genome and thereby possibly lead to clinical progression? § Can the IGHV-D-J sequences of the remaining non-CLL CD5+ B lymphocytes provide clues to the development of CLL from normal B cells?

Approach: § Sort all CD5 + cells from CLL pa2ent’s blood. This should yield the CLL clone, its intraclonal variants, and any normal CD5+ B cells leY in the blood § Perform high-throughput “deep” sequencing of the IGHV-D-J repertoire of FACS sorted CD19 + CD5 + from 39 untreated CLL pa2ents § Analyze data using appropriate bioinforma2c tools

Whole CD5 + B cell repertoire CLL clone

Whole CD5+ B cell repertoire CLL clone CLL sub-clones

CLL sub-clones developing through intraclonal diversifica2on can be significantly expanded CLL sub-clones CLL with most expanded sub-clone at > 1% 60% (n = 30) 100 > 1% < 1% % untreated 50 p=0.01 0 0 50 100 150 200 250 TFT (months)

CLL clones have evolved from precursors differing in IGHV muta2onal load Inferred Germline mline mline M- and U-CLL Clinically relevant CLL clone n=3 n= 1 mline mline 6 only M-CLL

Whole CD5 + B cell repertoire CLL clone CLL sub-clones

The CD19 + CD5 + compartment in CLL pa2ents contains leukemic and non-leukemic B-cell clones CLL with non-leukemic VDJ sequences > 0.75% Whole CD5+ B cell repertoire 45% (n = 22) CLL clone 100 > 0.75% < 0.75% % untreated 50 p=0.02 0 0 50 100 150 200 250 TFT (months)

Whole CD5 + B cell repertoire CLL clone CLL clone CLL sub-clones

CD19 + CD5 + B-cell popula2on can present an a second MBL-like clonal expansion Whole CD5+ B cell repertoire CLL with most expanded non-CLL clone at > 1% 24% (n = 12) CLL clone high-count MBL 19% (n=6) 1000 500 cells/ul low-count MBL 100 42% (n=12) 10 1 Rela2ve fitness Absolute fitness

Whole CD5+ B cell repertoire CLL clone CLL clone Non-leukemic clones CLL sub-clones

CD19 + CD5 + non-leukemic B-cell popula2on can be oligoclonally expanded CLL with 3 clones cons2tu2ng > 50% of the non-leukemic sequences 67% (n = 31) Non-leukemic clones

CLL present stereotyped IGHV-D-J sequences (Agathangelidis et al. Blood, 2012)

CLL-like stereotyped IGHV-D-J sequences can oYen be found among the non-leukemic clones in the CD19 + CD5 + compartment Leukemic 0.12 Non leukemic clones Non-leukemic clones clone CLL CLL samples CLL major subsets (n=50) 1 1 0.09 2 1 % of stereotyped clones Sample with Samples with 1 3 stereotyped stereotyped (n=2) 0.06 non-leukemic CLL clone 201 4 Clones (n=6) (n=6) 2 2 5 12% 12% 1 2 (x7), 3 , 5 , 14 (x2), 6 0.03 64B (x2) (Agathangelidis et al. Blood, 2012) 2 7 8 64B 0.00 1 (x5), 8 (IgG, UM) 9 Non − CLL clones Memory Memory − IgD Naive Transitional 4 (IgG, M), 64B 10

Implica2ons In > 80% of M-CLL pa2ents, IGHV-D-J sequence with lower muta2on load can be detected The leukemogenic process might start in a cell with less IGHV soma2c muta2ons

Implica2ons • Non-leukemic CD5 + B cells can display MBL-like oligoclonal expansion and CLL stereotypes - MBL-like oligoclonal origin of CLL - Genomic muta2onal events can occur at two stages of B-cell matura2on: - prior to IGHV-D-J recombina3on: ini2a2ng muta2on. (contribu2on of inherited factors?) - a`er IGHV-D-J recombina3on: final leukemogenic muta2on • Do not recapitulate typical CLL VH genes usage and HCDR3 length distribu2on - The final leukemogenic hint involve a further BCR selec2on

Implica2ons IGHV-D-J repertoire of CD5 + B cells can be clinically relevant: - Expanded CLL subclones might be a proxy of AID off-target muta2ons ac2vity and thereby indicate the poten2al genera2on of aggressive sub-clonal variants - Abundance of CD5 + non-leukemic sequences directly correlates with bemer outcome

Acknowledgments UNIVERSITÀ Davide Bagnara DEGLI STUDI DI GENOVA Stefano Vergani Andrea Mazzarello Gerardo Ferrer Anthony Liew

CD19 + CD5 + non-leukemic B-cell popula2on can be oligoclonally expanded Most expanded clone > 25% of the non-leukemic sequences 65% (n = 30) Non-leukemic clones

CD19 + CD5 + non-leukemic B-cell popula2on can be oligoclonally expanded Non-leukemic clones

Non-leukemic clones in the CD19 + CD5 + compartment do not present VH genes usage and HCDR3 length distribu2on typical of CLL CLL IgM controls non − leukemic CD5+ clones Mutated sequences Mutated Sequences Mutated Sequences Un − Mutated Sequences Un − Mutated Sequences SUBSET Un − Mutated sequences 0.25 0.25 CLL IgM controls non − leukemic CD5+ clones 0.10 0.20 0.20 0.15 0.15 Frequency Frequency Frequency 0.10 0.10 0.05 0.05 0.05 0.00 0.00 9 9 3 3 4 4 9 9 3 3 4 4 0.00 6 6 2 2 3 3 6 6 2 2 3 3 − − − − − − − − − − − − 1 1 3 3 4 4 1 1 3 3 4 4 V V V V V V V V V V V V H H H H H H H H H H H H 10 20 30 10 20 30 10 20 30 G G G G G G G G G G G G HCDR3 aa length I I I I I I I I I I I I VH genes VH genes