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.

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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.

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Supplemental material Baliakas et al

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 TP53abcases 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
Before first treatment / 1900/2995 (63%)
2326/2552(91%)
SF3B1 / Total:2322
≤12m from diagnosis
Before first treatment / 1421/2221 (64%)
1931/2051 (94%)
TP53 / Total:2309
≤12m from diagnosis
Before first treatment / 1387/2309 (66%)
1782/1923 (77%)
MYD88 / Total:1080
≤12m from diagnosis
Before first treatment / 843/1063 (79%)
951/1023 (93%)
BIRC3 / Total:919
≤12m from diagnosis
Before first treatment / 813/915 (89%)
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%)
BinetA / 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 (median) / 64 / 64 / 64 / 65 / 66

Supplemental Table 5. Overview of the methodological strategies employed by the collaborating institutions for the detection of mutationsin 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 sequencing, exons 4-10 (n=5/48) / Sanger sequencing, exon 34
Allele specific PCR for del7544-45(n=7/177)
Barcelona / Sanger sequencing, exons 4-9 (n=9/101)
NGS (n=7/196) / Sanger sequencing,
exon 34 (n=38/503) / Sanger sequencing, exons 14-16 (n=15/240)
NGS (n=17/184)
Brno / FASAY + Sanger sequencing, exons 4-10 (n=38/313) / Sanger sequencing, exons 6-9 (n=16/283) / Sanger sequencing, exon 5 (n=4/303) / Sanger sequencing,
del7544-45(n=41/304) / Sanger sequencing, exons 14-16 (n=43/304)
Milan / Sanger sequencing, exon 5 (n=1/97) / Sanger sequencing,
exon 34
Allele specific PCR for del7544-45 (n=12/133)
Novara / Sanger sequencing, exons 4-9 (n=30/402) / Sanger sequencing, exons 6-9 (n=7/402) / Sanger sequencing, exons 3 and 5 (n=12/402) / Sanger sequencing,
exon 34 (n=43/402) / Sanger sequencing, exons 14-16 (n=27/402)
Southampton/ Bournemouth / SSCP + Sanger sequencing, exons 5-8 (n=25/438) / Sanger sequencing,
exon 34, del7544-45
HRM analysis (n=60/694) / Sanger sequencing, exons 14-16
HRM analysis (n=111/661)
Thessaloniki / FASAY + Sanger sequencing, exons 4-10 (n=47/254) / Sanger sequencing,
exon 34
Allele specific PCR for del7544-45 (n=26/590)
Ulm / DHPLC + Sanger sequencing, exons 4-10 (n=62/180) / Sanger sequencing,
del7544-45 (n=22/171) / DHPLC
Sanger sequencing, exons 14-16 (n=35/171)
Uppsala / Sanger sequencing, exons 4-8 (n=14/361) / Sanger sequencing, exons 6-9 (n=0/234) / Sanger sequencing, exon 5, L265P
(n=7/268) / Sanger sequencing,
del7544-45 (n=17/360) / Sanger sequencing, exons 14-16 (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 mutations / BIRC3
frameshift deletions / BIRC3
ins/dup/SNV / FISH
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, c.1350dupT / del(11q)
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%)
MutantSF3B1 / 51/889 (5.7%)
MutantTP53 / 50/889 (5.6%)
MutantMYD88 / 15/628 (2.3%)
MutantBIRC3 / 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
Male
Female / 883 (346)
509 (210)
374 (136) / 8.1
9.9 / 0.088
Age
<55 years
>71 years / 178 (80)
250 (74) / 9.1
9 / ns
ns
IGHV
Mutated
Unmutated / 862 (338)
539 (123)
323 (215) / NR
3.25 / <0.0001
del(11q)
Positive
Negative / 823 (324)
95 (66)
728 (258) / 2.7
9.9 / <0.0001
del(13q)
Positive
Negative / 816 (320)
333 (102)
483 (218) / 11.8
5.5 / <0.0001
Trisomy 12
Positive
Negative / 832 (326)
124 (66)
708 (260) / 4.1
9.6 / <0.0001
MYD88
Mutated
WT / 624 (325)
15 (6)
609 (229) / NR
8.1 / ns
NOTCH1
Mutated
WT / 883 (346)
67 (46)
816 (300) / 3.3
9.9 / <0.0001
TP53ab
Positive
Negative / 826 (324)
56 (33)
770 (291) / 2.9
8.8 / <0.0001
SF3B1
Mutated
WT / 883 (346)
51 (35)
832 (311) / 3.1
9.8 / <0.0001
BIRC3
Mutated
WT / 616 (232)
11 (9)
605 (223) / 2.7
8.2 / 0.0001

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 analysisfor 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 Figure3. 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) amongSF3B1-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).

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