Nicholas Chiorazzi The Feinstein Ins3tute for Medical Research Northwell Health Manhasset, NY

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1 A Somewhat Different View of the Gene3c Portrait of Chronic Lymphocy3c Leukemia Nicholas Chiorazzi The Feinstein Ins3tute for Medical Research Northwell Health Manhasset, NY

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

3 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 ac2va2oninduced deaminase (AID)

4 IG V-region gene muta3ons in CLL Soma2c muta2ons of IGHV genes divide s/ 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

5 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 s 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.

6 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?

7 Approach: Sort all CD5 + cells from CLL pa2ent s blood. This should yield the, 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

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

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

10 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) % untreated > 1% < 1% p= TFT (months)

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

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

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

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

15 CD19 + CD5 + B-cell popula2on can present an a second MBL-like clonal expansion Whole CD5+ B cell repertoire CLL with most expanded non- at > 1% 24% (n = 12) most expanded non-leukemic clone cells/ul Absolute fitness Non-leukemic clones high-count MBL 19% (n=6) low-count MBL 42% (n=12) Rela2ve fitness smaller clones not smaller clon not CLL clo

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

17 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

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

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

20 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

21 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

22 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

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

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

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

28 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 0.25 Mutated Sequences Un Mutated Sequences CLL Mutated sequences Un Mutated sequences IgM controls 0.20 non leukemic CD5+ clones 0.10 Frequency Frequency IGHV1 69 IGHV3 23 IGHV4 34 IGHV1 69 IGHV3 23 IGHV HCDR3 aa length