Recurrent Mutations Refine Prognosis in Chronic Lymphocytic Leukemia

Supplemental material Baliakas et al

RECURRENT MUTATIONS REFINE PROGNOSIS IN CHRONIC LYMPHOCYTIC LEUKEMIA

Baliakas et al

The supplemental material contains the following tables and figures:

Supplemental Tables 1-13

Supplemental Figures 1-5.

1 Supplemental material Baliakas et al

Supplemental Table 1. Overview of general practice cases for each center included in the study.

GR01: n, % GR02: n, % IT01: n, % IT02: n, % SP01: n, % SE01: n, % CZ01: n, % UK01: n, % U-CLL 273/627,44% 102/211,48% 53/113,47% 138/395,35% 177/421,42% 119/348,34% 213/312,68% 44/116,38% Binet A 426/543,78% 54/77,70% 89/106,84% 315/402,78% 429/521,82% 244/318,77% 225/304,74% 205/229,89% Male 416/626,66% 131/211,62% 81/131,62% 217/402,54% 314/526,60% 237/368,64% 206/314,66% 134/232,58% del(13q) 105/330,32% 45/96,47% 136/400,34% 109/293,37% 171/344,50% 106/305,35% 76/175,43% Trisomy 12 68/341,20% 9/96,9% 84/400,21% 54/334,16% 37/344,11% 37/306,12% 50/185,27% del(11q) 40/341,12% 14/97,14% 35/400,9% 40/309,13% 37/344,11% 68/306,22% 19/212,9% del(17p) 23/344,7% 9/96,9% 33/400,8% 18/316,6% 12/344,3.5% 24/305,8% 5/211,2.5% m-NOTCH1 26/590,4.5% 7/177,4% 12/133,9% 43/402,11% 38/503,7.5% 17/360,5% 41/304,13.5% 12/228,5% m-SF3B1 27/402,7% 32/424,8% 13/360,4% 43/304,14% 21/225,9% m-TP53 47/254,19% 5/48,10% 3/16,18% 30/402,7.5% 16/297,5% 14/361,4% 38/313,12% m-MYD88 1/97,1% 12/402,3% 7/268,2.5% 4/313,1.5% m-BIRC3 7/402,1.8% 0/234,0% 16/283,5%

GR01: Thessaloniki, GR02: Athens, IT01: Milan, IT02: Novara, SP01: Barcelona, SE01: Uppsala, CZ01: Brno, UK01: Bournemouth. GR01 and GR02 perform TP53 test only in cases with unmutated IGHV genes. A total of 897 cases were tested for all 5 genes.

Supplemental Table 2. Overview of clinical trial cases included in the study.

UK LRFCLL4 CLL2H CLL 3X U-CLL 255/409 (62%) 73/96 (76%) 73/77 (95%) Male 364/493 (73%) 75/103 (73%) 55/80 (69%) del(13q) 192/466 (41%) 25/103 (24%) 18/80 (22%) Trisomy 12 76/464 (16%) 12/103 (12%) 4/80 (5%) del(11q) 92/464 (20%) 23/103 (22%) 27/80 (34%) del(17p) 20/460 (4%) 32/103 (31%) 15/80 (19%) m-NOTCH1 48/466 (10%) 13/97 (13.5%) 9/74 (12%) m-SF3B1 90/436 (21%) 17/97 (18%) 18/74 (24%) m-TP53 25/438 (6%) 38/100 (38%) 24/80 (30%) Enrichment of TP53ab cases in CLL2H and CLL3X.

Supplemental Table 3. Timing of testing in relation to the dates of diagnosis and first treatment.

Mutation Tested cases NOTCH1 Total: 3334 ≤12m from diagnosis 1900/2995 (63%) Before first treatment 2326/2552(91%) SF3B1 Total: 2322 ≤12m from diagnosis 1421/2221 (64%) Before first treatment 1931/2051 (94%) TP53 Total: 2309 ≤12m from diagnosis 1387/2309 (66%) Before first treatment 1782/1923 (77%) MYD88 Total: 1080 ≤12m from diagnosis 843/1063 (79%) Before first treatment 951/1023 (93%) BIRC3 Total: 919 ≤12m from diagnosis 813/915 (89%) Before first treatment 863/882 (98%)

Supplemental Table 4. Main clinicobiological characteristics of patients tested for each mutation. Enrichment of U-CLL among cases tested for TP53 mutations.

Parameters NOTCH1, n=3334 SF3B1, n=2322 TP53, n=2309 MYD88, n=1080 BIRC3, n=919 U-CLL 1298/2790 (46%) 907/1842 (49%) 1177/2094 (56%) 478/1040 (46%) 411/897 (46%) Binet A 1951/2486 (78%) 1346/1733(78%) 1237/1665 (74%) 765/1000 (77%) 665/869 (77%) Binet B/C 535/2486 (22%) 387/1733 (22%) 428/1665 (26%) 235/1000 (23%) 204/869 (23%) del(13q) 924/2484 (37%) 791/2052 (39%) 741/1999 (37%) 396/1019 (39%) 334/888 (38%) Trisomy 12 423/2576 (16%) 342/2100 (16%) 304/2030 (15%) 161/1020 (16%) 148/889 (17%) del(11q) 386/2580 (15%) 326/2107 (15%) 330/2017 (16%) 139/1021 (14%) 121/889 (14%) del(17p) 182/2583 (7%) 144/2108 (7%) 159/2022 (8%) 72/1019 (7%) 59/888 (7%) Male 2134/3329 (64%) 1472/2320 (63%) 1502/2307 (65%) 654/1078 (61%) 550/918 (60%) Age, years 64 64 64 65 66 (median)

Supplemental Table 5. Overview of the methodological strategies employed by the collaborating institutions for the detection of mutations in the present study. The primer sequences and reaction conditions are available upon request. In brackets: number of positive cases.

TP53 BIRC3 MYD88 NOTCH1 SF3B1 Athens FASAY + Sanger Sanger sequencing, sequencing, exon 34

exons 4-10 Allele specific PCR for (n=5/48) del7544-45(n=7/177) Barcelona Sanger Sanger sequencing, Sanger sequencing, exon 34 (n=38/503) sequencing, exons 4-9 exons 14-16 (n=9/101) (n=15/240) NGS (n=7/196) NGS (n=17/184) Brno FASAY + Sanger Sanger Sanger Sanger sequencing, Sanger sequencing, sequencing, sequencing, del7544-45 sequencing, exons 4-10 exons 6-9 exon 5 (n=41/304) exons 14-16 (n=38/313) (n=16/283) (n=4/303) (n=43/304) Milan Sanger Sanger sequencing, sequencing, exon 34 exon 5 (n=1/97) Allele specific PCR for del7544-45 (n=12/133) Novara Sanger Sanger Sanger Sanger sequencing, Sanger sequencing, sequencing, sequencing, exon 34 (n=43/402) sequencing, exons 4-9 exons 6-9 exons 3 and 5 exons 14-16 (n=30/402) (n=7/402) (n=12/402) (n=27/402) Southampton/ SSCP + Sanger Sanger sequencing, Sanger Bournemouth sequencing, exon 34, del7544-45 sequencing, exons 5-8 HRM analysis exons 14-16 (n=25/438) (n=60/694) HRM analysis (n=111/661) Thessaloniki FASAY + Sanger Sanger sequencing, sequencing, exon 34 exons 4-10 Allele specific PCR for (n=47/254) del7544-45 (n=26/590) Ulm DHPLC + Sanger Sanger sequencing, DHPLC sequencing, del7544-45 Sanger exons 4-10 (n=22/171) sequencing, (n=62/180) exons 14-16 (n=35/171) Uppsala Sanger Sanger Sanger Sanger sequencing, Sanger sequencing, sequencing, sequencing, del7544-45 sequencing, exons 4-8 exons 6-9 exon 5, L265P (n=17/360) exons 14-16 (n=14/361) (n=0/234) (n=7/268) (n=13/360) FASAY: functional analysis of separated allele in yeast; HPLC: high performance liquid chromatography; HRM: high resolution melting. NGS: next generation sequencing; SSCP: single strand conformational polymorphism.

Supplemental Table 6. Overview of the association of mutations in individual genes with clinicobiological characteristics.

NOTCH1 Mutated wild type p-value U-CLL 200/228, (88%) 1098/2562, (43%) <0.0001 Binet A 124/195, (64%) 1827/2291, (80%) <0.0001 Isolated del(13q) 60/196, (30%) 864/2288, (38%) 0.046 Trisomy 12 75/228, (33%) 348/2348, (15%) <0.0001 del(11q) 27/225, (12%) 359/2355, (15%) ns del(17p) 23/225, (10%) 159/2358, (7%) 0.06

SF3B1 Mutated wild type p-value U-CLL 148/202, (73%) 759/1640, (46%) <0.0001 Binet A 79/149, (53%) 1267/1584, (80%) <0.0001 del(13q) 78/243, (32%) 713/1809, (39%) 0.02 Trisomy 12 15/245, (6%) 327/1855, (17%) <0.0001 del(11q) 67/245, (27.3%) 259/1862, (14%) <0.0001 del(17p) 14/246, (5.7%) 130/1862, (7%) ns

TP53 Mutated wild type p-value U-CLL 151/205, (74%) 1026/1889, (54%) <0.0001 Binet A 104/175, (59%) 1133/1490, (76%) <0.0001 del(13q) 37/203, (18%) 704/1796, (39%) <0.0001 Trisomy 12 15/206, (7%) 289/1824, (16%) <0.0001 del(11q) 16/203, (8%) 314/1814, (17%) 0.0001 del(17p) 165/207, (80%) 41/1815, (2.2%) <0.0001

MYD88 Mutated wild type p-value U-CLL 0/23, (0%) 478/1017, (47%) <0.0001 Binet A 16/22, (73%) 749/978, (77%) ns del(13q) 13/22, (59%) 383/997, (38%) 0.05 Trisomy 12 1/22, (4.5%) 160/998, (16%) ns del(11q) 0/22, (0%) 139/999, (14%) 0.06 del(17p) 0/22, (0%) 72/997, (7%) ns

BIRC3 Mutated wild type p-value U-CLL 18/23, (78%) 393/874, (45%) 0.0001 Binet A 13/22, (59%) 652/847, (77%) 0.05 del(13q) 2/23, (9%) 332/865, (38%) 0.003 Trisomy 12 10/23, (43%) 138/866, (16%) 0.0004 del(11q) 11/23, (48%) 110/866, (13%) <0.0001 del(17p) 0/23, (0%) 59/865, (7%) ns

Supplemental Table 7. Clinicobiological profile of SF3B1 mutated cases harboring the p.K700E versus other mutations.

p.K700E, n=104 non-K700E, n=157 p-value U-CLL 50/83 (60%) 105/128 (82%) 0.009 del(13q) 39/100 (39%) 38/133 (29%) ns Trisomy 12 2/100 (2%) 13/144 (9%) 0.04 del(11q) 25/100 (25%) 41/144 (28%) ns del(17p) 4/100 (4%) 10/145 (6.9%) ns Binet A 31/57 (54%) 48/92 (52%) ns

Supplemental Table 8. Overview of the cases carrying BIRC3 aberrations.

No of BIRC3 BIRC3 BIRC3 FISH mutations frameshift deletions ins/dup/SNV RESULT 1 p.L548fs, c.1643delT Trisomy 12 1 p.K547fs, c.1639delC Trisomy 12 1 p.I362fs, c.1085delT del(11q) 1 p.E433fs, c.1295dupG Trisomy 12 1 p.K532fs, c.1598_1615delinsGTGGGAAT del(11q) 1 p.R600G, c.1798C>G del(11q) 1 p.?, c.1622-25_1622-5del del(13q) 1 p.C591Y, c. 1771G>A del(11q) 3 p.K547fs, c.1639delC p.C588S, c.1762T>A; p.R451X, del(11q) c.1350dupT 1 p.K532fs, c.1594_1598delAAATA Trisomy 12 1 p.L548fs, c.1643delT Trisomy 12 1 p.E553fs, c.1657_1666del del(11q) 1 p.S513fs, c.1538_1540delCTCinsT del(11q) 1 p.K547fs, c.1639delC all negative 1 p.Q552*, c.1664C>T del(11q) 1 p.V565fs, c.1692delA del(13q) 1 p.R555fs*12c.1663_1666del4 Trisomy 12 1 p.G367fs*6c.1101_1132del32 del(11q) 2 p.V395fs*78c.1183_1352del4894 p.E424*c.1270G>T del(11q) 1 p.Q547fs*12c.1638_1639insA Trisomy 12 1 p.R428fs*19c.1282delA del(11q)/trisomy 12 1 p.I427fs*11c.1279_1280insA Trisomy 12 1 p.I427fs*9c.1281_1285del5 Trisomy 12 Ins: insertion, dup: duplication, SNV: single nucleotide variation

Supplemental Table 9. Overview of the cases carrying 2 mutated genes.

SF3B1+TP53 SF3B1+NOTCH1 NOTCH1+TP53 n, (%) 31/1902 (1.6%) 15/2272 (0.6%) 28/2169 (1.2%) U-CLL 21/24 (88%) 10/13 (77%) 21/23 (91%) Binet A 9/21 (43%) 3/7 (43%) 12/23 (52%) Male 23/31 (74%) 12/15 (80%) 20/28 (71%) del(13q) 7/30 (23%) 0/14 (0%) 6/26 (23%) Trisomy12 0/30 (0%) 2/14 (14%) 5/26 (19%) del(11q) 5/30 (17%) 1/14 (7%) 1/26 (4%) del(17p) 9/31 (29%) 0/6 (0%) 12/26 (46%)

Co-existing mutations in the entire cohort: (i) concurrent NOTCH1 and TP53 mutations: 28/2169 (1.2%) cases; (ii) concurrent NOTCH1 and SF3B1 mutations: 15/2272 (0.6%) cases, with only 3/15 (20%) double mutant cases carrying the most prevalent p.K700E mutation; (iii) concurrent SF3B1 and TP53 mutations: 31/1902 (1.6%) cases; (iv) concurrent BIRC3 and NOTCH1 mutations: 4/906 (0.4%) cases; and, (v) concurrent BIRC3 and SF3B1 mutations: 2/909 (0.2%) cases. Only 2/1876 (0.1%) cases with available data harbored mutations in more than 2 of the 5 tested genes.

Supplemental Table 10. Main clinicobiological characteristics of Binet A cases included in the statistical analysis for time to first treatment. n (%) Male 514/889 (58%) U-CLL 323/868 (37%) del(13q) 336/821 (41%) del(11q) 96/828 (12%) Trisomy 12 124/838 (15%) del(17p) 37/832 (4.4%) Mutant NOTCH1 67/889 (7.5%) Mutant SF3B1 51/889 (5.7%) Mutant TP53 50/889 (5.6%) Mutant MYD88 15/628 (2.3%) Mutant BIRC3 11/620 (1.8%)

Origin of cases included in the multivariate analysis for time to first treatment: Czech Republic: 201, Spain: 194, Sweden: 179, Italy: 315

Supplemental Table 11. Univariate analysis for time to first treatment (TTFT)

Variable N (events) Median (years) p-value Gender 883 (346) 0.088 Male 509 (210) 8.1 Female 374 (136) 9.9 8 Supplemental material Baliakas et al

Age <55 years 178 (80) 9.1 ns >71 years 250 (74) 9 ns IGHV 862 (338) <0.0001 Mutated 539 (123) NR Unmutated 323 (215) 3.25 del(11q) 823 (324) <0.0001 Positive 95 (66) 2.7 Negative 728 (258) 9.9 del(13q) 816 (320) <0.0001 Positive 333 (102) 11.8 Negative 483 (218) 5.5 Trisomy 12 832 (326) <0.0001 Positive 124 (66) 4.1 Negative 708 (260) 9.6 MYD88 624 (325) ns Mutated 15 (6) NR WT 609 (229) 8.1 NOTCH1 883 (346) <0.0001 Mutated 67 (46) 3.3 WT 816 (300) 9.9 TP53ab 826 (324) <0.0001 Positive 56 (33) 2.9 Negative 770 (291) 8.8 SF3B1 883 (346) <0.0001 Mutated 51 (35) 3.1 WT 832 (311) 9.8 BIRC3 616 (232) 0.0001 Mutated 11 (9) 2.7 WT 605 (223) 8.2

Supplemental Table 12. Multivariate analysis for TTFT in U-CLL for Binet A cases.

HR 95%CI p-value TP53abn 2.356 1.473-3.770 0.000348 SF3B1 1.356 0.895-2.053 0.150091 male 1.521 1.125-2.055 0.006350 del(13q) 0.751 0.529-1.067 0.110474 del(11q) 1.228 0.884-1.706 0.220499

Supplemental Table 13. Multivariate analysis for overall survival (OS). Only variables significant in the univariate analysis have been included in the multivariate analysis.

HR 95%CI P NOTCH1 1.268 0.847-0.898 0.247 SF3B1 1.635 1.072-2.494 0.02 TP53ab 2.095 1.390-3.156 0.0004 U-CLL 2.319 1.764-3.050 <0.0001 del(13q) 0.976 0.753-1.267 0.9 del(11q) 1.101 0.761-1.592 0.6

Supplemental Figure 1. Hotspots of SF3B1 mutations.

Supplemental Figure 2. Concurrent mutations: TP53, SF3B1, NOTCH1.

Supplemental Figure 3. Survival curves for time-to-first-treatment. Effect of BIRC3 (A), TP53 (B), NOTCH1 (C) and SF3B1(D) mutations on Binet A cases.

Supplemental Figure 4. Survival curves for time-to-first-treatment. Effect of SF3B1 mutations on cases harboring del(13q) (A); NOTCH1 mutations on cases harboring trisomy 12 (B); TP53 mutations on cases harboring del(17p) (C); and type of mutation (p.K700E versus other) among SF3B1-mutant cases (D).

Supplemental Figure 5. Prognostic index proposed by Rossi et al for time-to-first-treatment (TTFT, 5A) and overall survival (OS, 5B) in Binet A patients of the present series. No difference between: high versus very high risk groups for TTFT (A); intermediate versus low risk groups or high versus very high risk groups for OS (B).