additional file 1 for implications for immune checkpoint

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 )


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 )


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 )


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 )


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 )


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 )


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


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 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 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


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


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


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

additional file 1 for implications for immune checkpoint

Documents