additional file 1 for implications for immune checkpoint
TRANSCRIPT
Additional file 1 for
Immunoglobulin somatic hypermutation has clinical impact in DLBCL and potential
implications for immune checkpoint blockade and neoantigen-based immunotherapies
Supplementary figure Legends
Figure S1. Construction and clinical outcome of the diffuse large B-cell
lymphoma (DLBCL) cohort. (a) CONSORT flow diagram illustrating the construction of the
training set and validation set comprising the overall study cohort. (b) Overall survival (OS) and
progression-free survival (PFS) rates of the overall patients included in this study, and OS/ PFS
comparison between germinal center B-cell-like (GCB) and activated B-cell-like (ABC) subtypes
of DLBCL.
Figure S2. Diagram showing the numbers of cases in this mutation study that
have been characterized by various biomarker studies, and survival rates of patients
whose sequencing results were correlated with prognosis.
Figure S3. CONSORT flow diagram illustrating the number of cases
performed for high-throughput IG sequencing and clonal sequence analysis. In the Venn
diagram, green number is for cases with only heavy chain analyzed, red number is for cases
with only light chain analyzed, and black number is for cases with both heavy and light chain
analyzed. In the flowchart, green boxes are for heavy chain analysis, and red boxes are for light
chain analysis.
Figure S4. Molecular characterization for immunoglobulin heavy chain
(IGH) gene usage in the study cohort. (a) Stacked histograms for IGHD gene usage in cases
with D-J-only (left) and cases with V-D-J (right) diagnostic sequences. (b-c) Stacked histograms
showing the specific IGHV gene usage pattern of diagnostic clones in overall DLBCL and
germinal-center B-cell–like (GCB) and activated B-cell–like (ABC) DLBCL subtypes. IGHV4-34
is over-represented in the ABC-DLBCL cases compared with the GCB-DLBCL cases.
Histogram showing the distribution of somatic mutations by V-gene family as measured by total
reads in GCB-DLBCL and ABC-DLBCL.
Figure S5. Immunoglobulin heavy chain V gene (IGHV) somatic
hypermutation (SHM) analysis. (a) Histogram showing the distribution of IGHV somatic
mutations within productive diagnostic clonal sequences. The number above the bar chart is the
absolute count of the total clones. GCB-DLBCL compared with ABC-DLBCL had a significantly
higher mean level of IGHV SHM. (b) High degree of IGHV SHM (SHMhigh) was associated with a
nonsignificant trend of better progression-free survival (PFS) in overall DLBCL. (c) IGHV
SHMhigh was associated with significantly better overall survival (OS) in ABC-DLBCL. (d) In
GCB-DLBCL, IGHV SHMhigh was associated with better OS with borderline significance only in
cases lacking BCL2 rearrangement (BCL2-R) or MYC rearrangement (MYC-R) but not in cases
with BCL2-R or MYC-R.
Figure S6. Analysis for length of heavy chain complementarity
determining region 3 (HCDR3). (a) Long length of HCDR3 with a cutoff of 18 amino acids (aa)
was associated with poorer progression-free survival (PFS) in DLBCL and ABC-DLBCL, and
with poorer PFS and overall survival (OS) in GCB-DLBCL. (b) Short HCDR3 length was
associated with significantly higher IGHV SHM degree in GCB-DLBCL, and higher IGK/L SHM
degree in ABC-DLBCL. The cutoff of short length was < 16 aa in GCB-DLBCL and <18 aa in
ABC-DLBCL. (c) Long HCDR3 length was associated with poorer OS in GCB-DLBCL cases of
the validation set and ABC-DLBCL cases of the training set. (d) Long HCDR3 length was
associated with significantly poorer PFS and OS in DLBCL in both the training and validation
sets.
Figure S7. Prediction of MHC-binding peptides and frequency of T-cell
exposed motifs (TCEM) for immunoglobulin diagnostic sequences in the training set and
validation set. (a) Regional distribution of relatively rare neoantigens (TCEM FC>16) derived
from heavy chain and light chain immunoglobulin genes in DLBCL patients in the training set
(top) and validation set (bottom). Each dot represents one peptide predicted having high MHC-
II-binding affinity (exceeding the -1 standard deviation threshold for MHC derived from 24 HLA-
DR alleles) and relatively rare TCEM (FC>16). The color intensities of the dots are scaled on
the FC scale from FC16 to the very rare FC24. (b) Cases with high degree of heavy chain or
light chain IGV SHM compared with cases without had higher frequency of relatively rare TCEM
(FC>16) in the training (left) and validation sets (right). (c) In ABC-DLBCL, high IGV SHM was
associated with lower tissue cellularity of CD4+ T cells.
Figure S8. Moleclar analysis for immunoglobulin heavy chain ongoing
SHM and light chain SHM. (a) Histogram showing distribution of IGHV ongoing SHM over
IGHV gene families. (b) High IGHV ongoing SHM was associated with AICDA upregulation in
the validation set. High PD-L2 expression in macrophages (in overall cohort) and PD-L1/PD-L2
gene amplification (in the ABC subtype) were associated with a higher mean percentage of
subclones with IGHV ongoing SHM in the sequence repertoire. (c) Histogram showing
distribution of IGK/LV SHM within light chain diagnostic clonal sequences. (d) Compared with
IGKV SHM, IGLV SHM showed more correlation with IGHV SHM.
Figure S9. Immunoglobulin light chain SHM and CDR3 analysis. (a)
IGK/LV SHMhigh was associated with significantly worse progression-free survival (PFS) in GCB-
DLBCL in both the training and validation sets. (b-c) High degree of IGK/LV SHM (SHMhigh) was
associated with significantly worse overall survival (OS) in GCB-DLBCL independent of BCL2
and MYC rearrangement (R) status. The P value was most significant in cases with BCL2
rearrangement. (d) A long immunoglobulin light chain kappa/lambda CDR3
(K/LCDR3) length was associated with poorer OS in overall DLBCL and ABC-DLBCL. (e) In
GCB-DLBCL, IGK/LV SHMhigh was significantly associated with higher CTSL1 mRNA levels
whereas IGHV SHMhigh was significantly associated with lower CTSF mRNA levels. (f) In ABC-
DLBCL, IGK/LV SHMhigh was negatively associated with PD-1 expression in B cells, and
positively associated with AICDA mRNA expression in the training set.
Figure S10. Comparison between different subsets of DLBCL. (a) The
training set compared with the validation set had significantly higher mean levels of some MHC
class II and cathepsin genes’ mRNA expression. (b) In the validation set overall (and the GCB
subtype, figures not shown), MYC rearrangement (MYC-R) was associated with downregulation
of some MHC class II genes. In both the training and validation sets, MYC-R was significantly
associated with HLA-F and CTSH downregulation (only figures for the entire cohort were
shown). (c) In both the training and validation sets, ABC-DLBCL compared with GCB-DLBCL
had significantly higher mean levels of macrophage- and CD8+ T cell-infiltration and PD-L1
expression on B cells whereas lower mean HLA-DQB2 mRNA level (only figures for overall
cohort were shown). In the overall cohort (and in the training set but not the validation set),
ABC- compared with GCB-DLBCL had significantly higher mean level of CTSL1 mRNA.
Figure S11. Light chain IGK/LV ongoing SHM analysis. (a) There were
negative associations between IGV SHM and light chain ongoing SHM. Like light chain SHMhigh,
(shown in Figure 5d) IGHV SHMhigh was associated with lower numbers of subclonal sequences
with IGK/LV ongoing SHM. Conversely, high numbers of IGK/LV subclonal sequences with
ongoing SHM were associated with lower mean levels of SHM in IGV. (b) Heatmap for gene
signatures associated with high ongoing IGK/LV SHM in DLBCL and GCB-DLBCL. (c) In GCB-
DLBCL, high IGK/LV ongoing SHM (≥17 subclones) was associated with significantly poorer
overall survival (OS). The adverse prognostic effect of light chain ongoing SHM was not
significant in the validation set. (d) In the training set, high light chain IGV ongoing SHM was
associated with significantly poorer survival.
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 1 6
PF
S (
%)
G C B (n = 2 0 2 )
A B C (n = 1 7 1 )
O v e ra ll D L B C L
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 1 1
OS
(%
)
G C B (n = 2 0 2 )
A B C (n = 1 7 1 )
O v e ra ll D L B C L
Training set (2014),n = 192
Validation set (2015),n= 186
Overall study cohort in this report, n = 378
Included patients,
NGS
de novo DLBCL,
NOS
Biopsy DNA
≥250ngTreatment: R-CHOP
BAFigure S1
Months294 cases by GEP;
79 cases by IHC algorithm
GCB, n = 202
ABC, n = 171
Excluded: PCNSL, cutaneous, HIV+, or transformed DLBCL
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
Su
rviv
al
(%)
O S (n = 3 7 8 )
P F S (n = 3 7 8 )
O v e ra ll D L B C L
IGK/Ln = 139
n = 85 n = 66
n = 290: index trackable sequences identified
n = 88: insufficient DNA sequencing reads or no expanded (>5%) clones
IG NGS n = 378
Figure S2
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 6
OS
(%
)
G C B (n = 1 4 6 )
A B C (n = 1 4 1 )
2 9 0 D L B C L
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 6 6
PF
S (
%)
G C B (n = 1 4 6 )
A B C (n = 1 4 1 )
2 9 0 D L B C L
Discard
Mutiplex IHC for cell type-specific PD-1/L1/2 expression, n = 230;FISH for PDL1, n = 235;FISH for PDL2, n = 234;FISH for MYC, n = 236; FISH for BCL2, n = 256;FISH for BCL6, n = 229;FISH for REL, n = 221;MYC mutation status, n = 268;BCL6 5ʹUTR mutation status, n = 151;TP53 mutation status, n = 263;IHC for IgM, IgA, and IgG expression, n = 280;IHC for Myc expression, n = 284;IHC for Bcl-2 expression, n = 281; IHC for CD37 expression, n = 273; IHC for CXCR4 expression, n = 258; IHC for CD30 expression, n = 284; IHC for CD5 expression, n = 283;IHC for p53 expression, n = 255;IHC for p63 expression, n = 274;IHC for MDM2 expression, n = 274;IHC for Ki-67 expression, n = 276; IHC for FOXP1 expression, n = 283; IHC for PI3K expression, n = 263;IHC for NF-ĸB p65 expression, n = 259;IHC for NF-ĸB p50 expression, n = 255;IHC for NF-ĸB p52 expression, n = 259
Months Months
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
Su
rviv
al
(%)
O S (n = 2 9 0 )
P F S (n = 2 9 0 )
2 9 0 D L B C L
246 cases by gene expression profiling (112 ABC, 117 GCB, 17 UC);
58 cases by IHC algorithm (29 GC, 29 non-GC)
GCB, n = 146
ABC, n = 141
Oth
er e
xper
imen
ts
IGH
IGH NGS n = 378
Expanded clones (index trackable
sequences), n = 224
No expanded clonal sequences,
n = 65
Reads insufficient for clonal analysis,
n = 89
Only D-J resolved, V unsolved, n = 79
Diagnostic IGHV resolved, n = 145
IGK/L NGSn = 269
No expanded clonal sequences,
n = 37
Expanded clones (index trackable sequences),
n = 205
Reads insufficient for clonal analysis,
n = 27
Unproductive, n = 42
Productive, n = 163
Figure S3
Productive, n = 138
Unproductive, n = 7
IGK/L
n = 85 n = 66
290 cases with index trackable sequences identified
n = 139IGH
IGHD1
IGHD2
IGHD3
IGHD4
IGHD5
0%
5%
10%
15%
20%
25%
IGHD1
IGHD2
IGHD3
IGHD4
IGHD5
IGHD6
IGHD7
0%
5%
10%
15%
20%
25%
30%
35%
Cases with only-D-J (V-unresolved) diagnostic sequences
30%
Cases with V-D-J diagnostic sequences40%
% o
f tot
al c
ases
IGHD6
02
30 34
69
4 61
23
0%
10%
20%
30%
40%
50%
60%
IGHV
01
IGHV
02
IGHV
03
IGHV
04
IGHV
07
IGHV
_III_
OR1
6
69
48
3402
2
61
53
0%
10%
20%
30%
40%
50%
60%
IGHV
01
IGHV
02
IGHV
03
IGHV
04
IGHV
05
DLBCL
ABC-DLBCLIGHV1
IGHV2
IGHV3
IGHV4
IGHV5
0%
10%
20%
30%
40%
55%
50%
IGHV7
IGHVIII
IGHV1
IGHV2
IGHV3
IGHV4
IGHV7
IGHVIII
0%
10%
20%
30%
40%
60%
50%
0%
% o
f tot
al c
ases
10%
20%
30%
40%
60%
50%
GCB-DLBCL
5%
15%
25%
35%
45%
IGHV1
IGHV2
IGHV3
IGHV4
IGHV5
C
AFigure S4
B
IGH
V s
om
ati
c m
uta
tio
n (
%)
G C B A B C0
5
1 0
1 5
2 0
2 5P = 0 .0 1 2
n = 6 6 n = 7 7
A
Figure S5
B
Months
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 5
OS
(%
)
IG H V S H M lo w (n = 4 3 )
IG H V S H M h ig h (n = 3 4 )
A B C -D L B C L
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 5 9
OS
(%
)
IG H V S H M lo w (n = 1 8 )
IG H V S H M h ig h (n = 1 5 )
B C L 2 -R ¯ G C B -D L B C LD
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 6 6
OS
(%
)
IG H V S H M lo w (n = 1 6 )
IG H V S H M h ig h (n = 2 5 )
M Y C -R ¯ G C B -D L B C L
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .3 1
OS
(%
)
IG H V S H M lo w (n = 4 )
IG H V S H M h ig h (n = 4 )
M Y C -R + G C B -D L B C L
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .8 6
OS
(%
)
IG H V S H M lo w (n = 6 )
IG H V S H M h ig h (n = 1 8 )
B C L 2 -R + G C B -D L B C L
Months Months Months Months
C
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .1 0
PF
S (
%) IG H V S H M lo w (n = 7 2 )
IG H V S H M h ig h (n = 7 3 )
D L B C L
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 5 4
PF
S (
%)
H C D R 3 le n g th ≤ 1 7 a a (n = 4 2 )
H C D R 3 le n g th ≥ 1 8 a a (n = 3 0 )
A B C -D L B C LA
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 0 2
PF
S (
%)
H C D R 3 le n g th ≤ 1 7 a a (n = 7 7 )
H C D R 3 le n g th ≥ 1 8 a a (n = 5 9 )
D L B C L
CB
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 1 6
OS
(%
)
H C D R 3 le n g th ≤ 1 7 a a (n = 3 4 )
H C D R 3 le n g th ≥ 1 8 a a (n = 2 9 )
G C B -D L B C L
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 1 5
PF
S (
%)
H C D R 3 le n g th ≤ 1 7 a a (n = 3 4 )
H C D R 3 le n g th ≥ 1 8 a a (n = 2 9 )
G C B -D L B C L
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 5 5
OS
(%
)
H C D R 3 s h o rt (n = 2 2 )
H C D R 3 lo n g (n = 1 6 )
A B C -D L B C LABC-DLBCL, training set
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 0 9 3
OS
(%
)
H C D R 3 s h o rt (n = 1 5 )
H C D R 3 lo n g (n = 2 0 )
G C B -D L B C LGCB-DLBCL, validation set
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 4 4
OS
(%
)
H C D R 3 s h o rt (n = 3 7 )
H C D R 3 lo n g (n = 2 9 )
D L B C L
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 3 6
PF
S (
%)
H C D R 3 s h o rt (n = 3 7 )
H C D R 3 lo n g (n = 2 9 )
D L B C LDLBCL, training set DLBCL, training set
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 3 3
OS
(%
)
H C D R 3 s h o rt (n = 3 6 )
H C D R 3 lo n g (n = 3 4 )
D L B C L
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 1 2
PF
S (
%)
H C D R 3 s h o rt (n = 3 6 )
H C D R 3 lo n g (n = 3 4 )
D L B C LDLBCL, validation setDLBCL, validation set
Figure S6
Months Months Months Months
D
IGK
/LV
so
ma
tic
mu
tati
on
(%
)
H C D R 3 s h o r t H C D R 3 lo n g-5
0
5
1 0
1 5 P = 0 .0 2 3
n = 2 7 n = 2 2
A B C -D L B C L
IGH
V s
om
ati
c m
uta
tio
n (
%)
H C D R 3 s h o r t H C D R 3 lo n g0
5
1 0
1 5
2 0
2 5 P = 0 .0 3 2
n = 3 0 n = 3 1
G C B -D L B C L
Figure S7
CD
4+
T c
ell
ce
llu
lari
ty (
%)
IG V H S H M lo IG V H S H M h i-2 0
0
2 0
4 0
6 0
8 0
P = 0 .0 3 2
n = 3 4 n = 2 5
A B C -D L B C L
A B
C
CD
4+
T c
ell
ula
rity
(%
)
IG V K /L S H M lo w IG V K /L S H M h ig h-2 0
0
2 0
4 0
6 0
8 0
1 0 0P = 0 .0 3 2
n = 6 7 n = 1 3
A B C -D L B C L
Fre
qu
en
cy
of
su
bc
lon
es
wit
hIG
HV
on
go
ing
SH
M (
%)
P D L 1 A m p lif¯ P D L 1 A m p lif+-2 0
0
2 0
4 0
6 0
P = 0 .0 0 2 8
n = 5 4 n = 3
A B C -D L B C L
Fre
qu
en
cy
of
su
bc
lon
es
wit
hIG
HV
on
go
ing
SH
M (
%)
P D L 2 A m p lif¯ P D L 2 A m p lif+-2 0
0
2 0
4 0
6 0
P = 0 .0 1 4
n = 5 3 n = 4
A B C -D L B C L
Figure S8
05
1015202530354045
IGHV_III IGHV01 IGHV02 IGHV03 IGHV04 IGHV05 IGHV06 IGHV07
Ongoing SHM by V family
No Ongoing Ongoing <10 sequences Ongoing >=10 sequences
Num
ber o
f cas
es
A B
CD
IGK
/LV%
som
atic
mut
atio
n
IGHV% somatic mutation
IGH and IGL pair
IGH and IGK pair
Fre
qu
en
cy
of
su
bc
lon
es
wit
hIG
HV
on
go
ing
SH
M (
%)
P D -L 2 ¯ C D 6 8 + P D -L 2 + C D 6 8 +-2 0
0
2 0
4 0
6 0
P = 0 .0 0 7 4
n = 8 9 n = 4
AIC
DA
mR
NA
(L
og
2)
S H M o n g o in g ¯ S H M o n g o in g +0
5
1 0
1 5P = 0 .0 0 0 2
n = 3 1 n = 1 8
V a lid a tio n s e t
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 2 3
PF
S (
%)
IG K /L V S H M lo w (n = 5 3 )
IG K /L V S H M h ig h (n = 1 4 )
G C B -D L B C L
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 2 2
PF
S (
%)
IG K /L V S H M lo w (n = 2 5 )
IG K /L V S H M h ig h (n = 7 )
G C B -D L B C L
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
OS
(%
)
P = 0 .0 2 3
B C L 2 -R ¯ G C B -D L B C L
IG K /L V S H M h ig h (n = 8 )
IG K /L V S H M lo w (n = 4 7 )
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
OS
(%
)
P = 0 .0 0 1 4
B C L 2 -R + G C B -D L B C L
IG K /L V S H M h ig h (n = 1 1 )
IG K /L V S H M lo w (n = 2 4 )
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
OS
(%
)
P = 0 .0 1 6
M Y C -R ¯ G C B -D L B C L
IG K /L V S H M h ig h (n = 1 5 )
IG K /L V S H M lo w (n = 5 9 )
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
OS
(%
)
P = 0 .0 1 9
M Y C -R + G C B -D L B C L
IG K /L V S H M h ig h (n = 3 )
IG K /L V S H M lo w (n = 7 )
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 2 6
OS
(%
)
K /L C D R 3 le n g th ≤ 1 2 a a (n = 1 6 0 )
K /L C D R 3 le n g th ≥ 1 3 a a (n = 1 2 )
D L B C L
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 1 2
OS
(%
)
K /L C D R 3 le n g th ≤ 1 2 a a (n = 8 4 )
K /L C D R 3 le n g th ≥ 1 3 a a (n = 7 )
A B C -D L B C L
A
C D
Months Months Months Months
E F
CT
SL
1 m
RN
A (
Lo
g2
)
IG V K /L S H M lo w IG V K /L S H M h ig h6
7
8
9
1 0
1 1
1 2P = 0 .0 3 8
n = 5 8 n = 1 9
G C B -D L B C L
CT
SF
mR
NA
(L
og
2)
IG V H S H M lo w IG V H S H M h ig h5
6
7
8
9P = 0 .0 4 8
n = 2 1 n = 3 3
G C B -D L B C L
PD
-1+
CD
20
+/C
D2
0+
(%)
IG V K /L S H M lo w IG V K /L S H M h ig h-2 0
0
2 0
4 0
6 0
8 0
1 0 0P = 0 .0 3
n = 6 7 n = 1 2
A B C -D L B C L
AIC
DA
mR
NA
(L
og
2)
IG V K /L S H M lo IG V K /L S H M h i0
5
1 0
1 5P = 0 .0 4 7
n = 1 0 6 n = 2 7
T ra in in g s e t A B C -D L B C L
Figure S9GCB-DLBCL, training set GCB-DLBCL, validation set B
CT
SH
mR
NA
(lo
g2
)
M Y C -R ¯ M Y C -R +6
8
1 0
1 2
P < 0 .0 0 0 1
n = 2 1 9 n = 2 8
HL
A-F
mR
NA
(Lo
g2
)
M Y C -R ¯ M Y C -R +3
4
5
6
7
8P = 0 .0 0 0 2
n = 8 9 n = 1 2
G C B -D L B C L
CT
SH
mR
NA
(lo
g2
)
T r a in in g V a l id a t io n
6
8
1 0
1 2
P < 0 .0 0 0 1
n = 1 8 3 n = 1 3 5
AFigure S10
HL
A-D
RB
1/4
mR
NA
(L
og
2)
t r a in in g v a l id a t io n6
8
1 0
1 2
1 4P = 0 .0 0 1 5
n = 1 8 3 n = 1 3 5
HL
A-D
RA
mR
NA
(L
og
2)
t r a in in g v a l id a t io n5
1 0
1 5P = 0 .0 0 2 8
n = 1 8 3 n = 1 3 5
HL
A-D
PB
1 m
RN
A (
Lo
g2
)
t r a in in g v a l id a t io n
6
8
1 0
1 2P = 0 .0 0 1 3
n = 1 8 3 n = 1 3 5
HL
A-D
PA
1 m
RN
A (
Lo
g2
)
t r a in in g v a l id a t io n
6
8
1 0
1 2P = 0 .0 0 3 7
n = 1 8 3 n = 1 3 5
HL
A-D
RB
1/4
RN
A (
log
2)
M Y C -R ¯ M Y C -R +6
8
1 0
1 2
1 4
P = 0 .0 0 3 4
n = 6 1 n = 8
V a lid a t io n s e t
HL
A-D
PB
1R
NA
(lo
g2
)
M Y C -R ¯ M Y C -R +
6
8
1 0
1 2
P = 0 .0 0 2
n = 6 1 n = 8
V a lid a t io n s e t
HL
A-D
PA
1R
NA
(lo
g2
)
M Y C -R ¯ M Y C -R +
6
8
1 0
1 2
P = 0 .0 0 1 7
n = 6 1 n = 8
V a lid a t io n s e t
HL
A-D
QB
2 m
RN
A (
Lo
g2
)
G C B A B C0
5
1 0
1 5P < 0 .0 0 0 1
n = 1 6 8 n = 1 4 9
CT
SL
1 m
RN
A (
log
2)
G C B A B C
6
8
1 0
1 2
P = 0 .0 0 0 1
n = 1 6 8 n = 1 4 9
CD
8+
T c
ell
in
filt
rati
on
(%
)
G C B A B C0
2 0
4 0
6 0
8 0P = 0 .0 0 4 4
n = 1 6 1 n = 1 3 9
Ma
cro
ph
ag
e i
nfi
ltra
tio
n (
%)
G C B A B C0
1 0
2 0
3 0
4 0
5 0P = 0 .0 0 1 4
n = 1 6 1 n = 1 3 9
B
PD
-L1
+C
D2
0+
/CD
20
+ (
%)
G C B A B C-2 0
0
2 0
4 0
6 0
8 0
1 0 0P = 0 .0 0 0 1
n = 1 5 6 n = 1 3 7
C
IGK
/LV
SH
M (
%)
IG V K /L S H M o n g o in glo IG V K /L S H M o n g o in g
h i0
5
1 0
1 5
2 0
2 5P = 0 .0 3 1
n = 9 4 n = 9
IGH
V S
HM
(%
)
IG V K /L S H M o n g o in glo IG V K /L S H M o n g o in g
h i0
5
1 0
1 5
2 0
2 5P = 0 .0 0 1 9
n = 3 3 n = 6
No
. o
f s
eq
ue
nc
es
wit
hIG
K/L
V o
ng
oin
g S
HM
IG V H S H M lo IG V H S H M h i-2 0
0
2 0
4 0
6 0
8 0
1 0 0P = 0 .0 2 3
n = 1 6 n = 2 3
DLBCL, IGK/LV ongoing SHMlow vs. ongoing SHMhigh
Figure S11
GCB-DLBCL, IGK/LV ongoing SHMlow vs. ongoing SHMhigh
A B
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 0 2 2
OS
(%
)
D L B C L
0 -1 6 IG K /L V s e q u e n c e sw ith o n g o in g S H M (n = 6 6 )
≥ 1 7 IG K /L V s e q u e n c e sw ith o n g o in g S H M (n = 6 )
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 0 1
PF
S (
%)
D L B C L
0 -1 6 IG K /L s e q u e n c e s w itho n g o in g S H M (n = 6 6 )
≥ 1 7 IG K /L V s e q u e n c e s w itho n g o in g S H M (n = 6 )
DLBCL, training set DLBCL, training set
Months Months
C
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .4 6
OS
(%
)
D L B C L
0 -1 6 IG K /L s e q u e n c e s w itho n g o in g S H M (n = 9 )
≥ 1 7 IG K /L s e q u e n c e s w itho n g o in g S H M (n = 3 )
DLBCL, Validation set
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 00
2 0
4 0
6 0
8 0
1 0 0
P = 0 .0 4 2
OS
(%
)
O n g o in g IG K /L S H M lo w (n = 4 5 )
O n g o in g IG K /L S H M h ig h (n = 8 )
G C B -D L B C LGCB-DLBCL (overall)
D