Published Ahead of Print on March 6, 2015, as doi: /haematol Copyright 2015 Ferrata Storti Foundation.

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1 Published Ahead of Print on March 6, 2015, as doi: /haematol Copyright 2015 Ferrata Storti Foundation. Somatic mutations of cell-free circulating DNA detected by Next Generation Sequencing reflect the genetic changes in both Germinal Center B-Cell like and Activated B-Cell like Diffuse Large B-Cell Lymphoma tumors at the time of diagnosis by Elodie Bohers, Pierre Julien Viaillly, Sydney Dubois, Philippe Bertrand, Catherine Maingonnat, Sylvain Mareschal, Philippe Ruminy, Jean-Michel Picquenot, Christian Bastard, Fabienne Desmots, Thierry Fest, Karen Leroy, Hervé Tilly, and Fabrice Jardin Haematologica 2015 [Epub ahead of print] Citation: Bohers E, Viaillly PJ, Dubois S, Bertrand P, Maingonnat C, Mareschal S, Ruminy P, Picquenot JM, Bastard C, Desmots F, Fest T, Leroy K, Tilly H, and Jardin F. Somatic mutations of cell-free circulating DNA detected by Next Generation Sequencing reflect the genetic changes in both Germinal Center B-Cell like and Activated B-Cell like Diffuse Large B-Cell Lymphoma tumors at the time of diagnosis. Haematologica. 2015; 100:xxx doi: /haematol Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepted for publication. E-publishing of this PDF file has been approved by the authors. After having E-published Ahead of Print, manuscripts will then undergo technical and English editing, typesetting, proof correction and be presented for the authors' final approval; the final version of the manuscript will then appear in print on a regular issue of the journal. All legal disclaimers that apply to the journal also pertain to this production process.

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13 Supplementary file 1. Lymphopanel and PGM data analysis A. Lymphopanel set used for NGS experiments Gene Transcript Reference Hotspot / Exons partially sequenced Chromosomal location Size sequenced (bp) B2M NM_ Hotspots exons 1 & 2, exon 3 15q BCL2 NM_ Hotspot exon 2 18q BRAF NM_ Exon 15 7q CARD11 NM_ Coiled-coil domain exons 4-9 7p CD58 NM_ Exons 1-6 1p CD79A NM_ ITAM domain exons 4 & 5 19q CD79B NM_ ITAM domain exons 5 & 6 17q CDKN2A NM_ NM_ NM_ NM_ Exons 1, 2A, 2B, 3, 4 & 5 9p21.3 1,737 CDKN2B NM_ NM_ Exons 1A, 1B & 2 9p CIITA NM_ Exons p CREBBP NM_ Exons p EP300 NM_ Exons q EZH2 NM_ SET domain, hotspots exon 16 & 18 7q FOXO1 NM_ Hotspots exon 1 & FH domain exon 2 13q GNA13 NM_ Exons q ID3 NM_ Exons 1 & 2 1p IRF4/MUM1 NM_ Exons 2-9 6p ITPKB NM_ Exons 2-8 1q KMT2D/MLL2 NM_ Exons q MEF2B NM_ Exons p MFHAS1 NM_ Exons 1-3 8p MYC NM_ Exons 1-3 8q MYD88 NM_ Exons 2-5 3p NOTCH1 NM_ PEST domain exon 34 9q NOTCH2 NM_ Exons & 34 (HD/PEST domains) 1p12-p PIM1 NM_ Exons 1-6 6p PRDM1/BLIMP1 NM_ Exons 1-7 6q SOCS1 NM_ Exon 2 16p STAT6 NM_ Exons 9-14 (DNA binding domain hotspot) 12q TCF3 NM_ B-HLH domain of E47 isoform exons 17 & 18 19p TNFAIP3 NM_ Exons 2-9 6q TNFRSF14 NM_ Exons 1-8 1p TP53 NM_ Mutation hotspots exons p XPO1 NM_ Exons p15 640

14 B. PGM Data analysis Torrent Suite version 4.0 (Life Technologies) software was used to perform primary analysis, including signal processing, base calling, sequence alignment to the reference genome (hg19) and generation of Binary Alignment/Map (BAM) files. BAM files were used by Torrent Suite s Variant Caller to detect point mutations as well as short insertions and deletions using the Somatic and Low Stringency default parameters. VCF files generated by Variant Caller were annotated by ANNOVAR(1). Data generated from tumor DNA samples were considered of sufficient quality when more than 90% of targeted bases were read at least 20 times with sequencing and mapping precisions of at least Q20. Only frameshift deletions and insertions, nonframeshift deletions and substitutions, splicing, nonsynonymous, stopgain or stoploss Single Nucleotide Variations (SNVs) were kept. Variants with a minimal Variant Allele Frequency (MAF) greater than 1% in the 1000 genome database were considered as polymorphisms and were discarded(2). A normal probability plot defined thresholds separating true positives [confirmed by Sanger sequencing, TVC (Torrent variant calling) quality score 22] from true negatives (discredited by Sanger sequencing, TVC score < 9.5) and highlighted a gray zone (9.5 TVC score < 22) in which variants must be confirmed by Sanger sequencing or pyrosequencing.. Further verification by Sanger sequencing was performed using a BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) and an ABI PRISM 3130 analyzer (Life Technologies). Further verification by pyrosequencing was performed using the PyroMark PCR kit (Qiagen, France) with internal and sequencing primers designed using PyroMark software (Qiagen). For circulating DNA variant calling, the parameter file was modified as follows: hotspot_min_variant_score: 3; hotspot_strand_bias: 1; downsample_to_coverage: In addition, a dedicated hotspot VCF file generated from variants found in tumor DNA was used. This file instructs the Variant Caller to include these positions in its output files, including evidence for a variant and the filtering thresholds that disqualify a variant candidate. References 1. Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of genetic variants from highthroughput sequencing data. Nucleic acids research Sep;38(16):e Abecasis GR, Auton A, Brooks LD, DePristo MA, Durbin RM, Handsaker RE, et al. An integrated map of genetic variation from 1,092 human genomes. Nature Nov 1;491(7422):56-65.

15 Figure S1 Tumor: 885 Plasma: 21,078 Tumor: 582 Plasma: 18,827 Tumor: 553 Plasma: 4,685

16 Figure S1 Tumor: 1,263 Plasma: 21,661 Tumor: 1,381 Plasma: 51,195

17 Figure S1 Tumor: 1,886 Plasma: 70,466

18 Figure S1 Tumor: 3421 Plasma: 77,872 Tumor: 636 Plasma: 386

19 Figure S1 Tumor: 3,724 Plasma: 36,037 Tumor: 1,811 Plasma: 58,131

20 Figure S1 Tumor: 2,041 Plasma: 85,369 Tumor: 1,852 Plasma: 50,862

21 Supplementary table 1 UPN Sex Age Stage IPI VAF (%) read number mean VAF (%) VAF (%) read number circulating DNA mean VAF Allele Call (%) concentration (ng/µl) ITPKB NM_002221:exon2:c.C731T-(p.T244I) / /6313 Heterozygous ITPKB NM_002221:exon2:c.726_728del-(p.242_243del) / /6260 Heterozygous 964 F 17 IV GCB CARD11 NM_032415:exon7:c.G1010A-(p.R337Q) / / Absent EZH2 NM_004456:exon16:c.A1937T-(p.Y646F) / /4435 Heterozygous B2M NM_004048:exon1:c.T2A-(p.M1K) / /4016 Heterozygous 1251 M 42 IE 0 ITPKB NM_002221:exon2:c.541delC-(p.R181fs) / /1540 Heterozygous MYD88 NM_002468:exon4:c.T695C-(p.M232T) / /21 Absent ABC CIITA NM_000246:exon13:c.C2819A-(p.T940K) / /6 Heterozygous CD79B NM_000626:exon6:c.596delT-(p.L199fs) / /17260 Heterozygous TNFRSF14 NM_003820:exon1:c.G3A-(p.M1I) / /1113 Absent GCB EZH2 NM_004456:exon16:c.A1937T-(p.Y646F) / / Absent 0.46 SOCS1 NM_003745:exon2:c.C174A-(p.F58L) / /1053 Absent CD79A NM_001783:c.568-2A>G / /386 Heterozygous 1437 M 76 III ABC CD79A NM_001783:exon5:c573_617del-(p.191_206del) / /386 Heterozygous 1524 F 45 III F 66 III 4 LDH (x UNL) Bone marrow involvement 1003 F 75 III Phenotype Gene SNV Tumor DNA TNFRSF14 NM_003820:exon3:c.T269C-(p.L90P) / /3064 Heterozygous EZH2 NM_004456:exon16:c.T1936A-(p.Y646N) / /11751 Heterozygous KMT2D NM_003482:exon40:c.G13671C-(p.Q4557H) / /10509 Heterozygous KMT2D NM_003482:exon34:c.C8401T-(p.R2801X) / /6818 Heterozygous GCB B2M NM_004048:exon1:c.T35C-(p.L12P) / /4555 Absent CREBBP NM_004380:c A>T / /331 Heterozygous GNA13 NM_006572:exon3:c.T533A-(p.L178Q) / /8254 Heterozygous GNA13 NM_006572:exon3:c.T530C-(p.F177S) / /8314 Heterozygous GNA13 NM_006572:exon3:c.T526A-(p.Y176N) / /8312 Heterozygous BCL2 NM_000633:exon2:c.G17C-(p.R6T) / /8558 Heterozygous ITPKB NM_002221:exon2:c.C1583T-(p.S528L) / /1713 Absent ITPKB NM_002221:exon2:c.C1471G-(p.P491A) / /3582 Absent ITPKB NM_002221:exon2:c.C1454A-(p.A485E) / /3577 Absent ITPKB NM_002221:exon2:c.C1438G-(p.P480A) / /3570 Absent MYD88 NM_002468:exon4:c.G728A-(p.S243N) / /1888 Heterozygous TNFAIP3 NM_006290:exon9:c.T2335C-(p.C779R) / /3152 Heterozygous EZH2 NM_004456:exon16:c.T1936A-(p.Y646N) / /7401 Heterozygous KMT2D NM_003482:exon48:c.C15142T-(p.R5048C) / /3620 Heterozygous GCB B2M NM_004048:exon1:c.G3A-(p.M1I) / / Heterozygous 1.94 CIITA NM_000246:exon11:c.2239delT-(p.Y747fs) / /2130 Absent CIITA NM_000246:exon18:c.T3236A-(p.V1079D) / /9150 Absent CIITA NM_000246:exon18:c.T3241A-(p.Y1081N) / /9162 Absent GNA13 NM_006572:exon1:c.G201C-(p.Q67H) / /2695 Absent GNA13 NM_006572:exon1:c.T158C-(p.L53P) / /2672 Heterozygous BCL2 NM_000657:exon2:c.G589A-(p.G197S) / /4929 Heterozygous BCL2 NM_000633:exon2:c.C502G-(p.P168A) / /5013 Heterozygous MEF2B NM_ :exon3:c.G229C-(p.E77Q) / /11153 Heterozygous Duration of Tumor/plasma storage (y) before sequencing 10 7

22 Supplementary table 1 CD58 NM_001779:exon3:c.C454T-(p.R152X) / /13231 Absent MYD88 NM_002468:exon5:c.T778C-(p.L265P) / /11427 Absent 1559 F 83 IE ABC CREBBP NM_004380:exon30:c.G5105T-(p.R1702L) / /5663 Absent 64.0 <1 TP53 NM_000546:exon5:c.G524A-(p.R175H) / /13012 Absent CD79A NM_001783:c.568-2A>G / /3930 Absent CD79A NM_001783:exon5:c573_617del-(p.191_206del) / /3932 Absent 1586 F 62 III M 53 IV 2 TNFRSF14 NM_003820:exon2:c.C164T-(p.P55L) / /284 Heterozygous EZH2 NM_004456:exon16:c.A1937T-(p.Y646F) / /6575 Absent NA STAT6 NM_ :exon12:c.A1256G-(p.D419G) / /5517 <1 Absent CREBBP NM_004380:exon26:c.G4283C-(p.R1428P) / /4894 Absent GNA13 NM_006572:c.561+2A>G / /4391 Absent TNFRSF14 NM_003820:c.179-1G>C / /3587 Heterozygous EZH2 NM_004456:exon16:c.A1937T-(p.Y646F) / /3480 Heterozygous STAT6 NM_ :exon12:c.A1256G-(p.D419G) / /3889 Heterozygous GCB CREBBP NM_004380:exon28:c.A4628T-(p.D1543V) / / Heterozygous 0.95 CREBBP NM_004380:exon27:c.T4504C-(p.W1502R) / /3239 Heterozygous CD79B NM_000626:exon6:c.611dupC-(p.T204fs) / /4651 Heterozygous MEF2B NM_ :exon3:c.G229A-(p.E77K) / /12969 Heterozygous MYD88 NM_002468:exon4:c.G728A-(p.S243K) / /5586 Heterozygous PIM1 NM_002648:exon4:c.G290A-(p.S97N) / /4797 Heterozygous CARD11 NM_032415:exon5:c.645_647del-(p.215_216del) / /3028 Heterozygous 1631 M 64 I ABC CARD11 NM_032415:exon4:c.T343C-(p.F115L) / / Heterozygous STAT6 NM_ :exon12:c.A1249G-(p.N417D) / /13250 Heterozygous TP53 NM_000546:exon5:c.T518A-(p.V173E) / /13317 Heterozygous CD79B NM_000626:exon5:c.A587T-(p.Y196F) / /11853 Heterozygous 1639 M 69 IV TNFRSF14 NM_003820:exon6:c.C620A-(p.S207X) / /2915 Heterozygous MYD88 NM_002468:exon3:c.C840G-(p.S219C) / /22541 Heterozygous EZH2 M_004456:exon16:c.A1937T-(p.Y646F) / /9939 Heterozygous KMT2D NM_003482:exon34:c.C9838T-(p.Q3280X) / /16784 Heterozygous GCB KMT2D NM_003482:exon22:c.5296_5297insGAGG-(p.D1766fs) / / Heterozygous CREBBP NM_004380:c A>G / /9530 Heterozygous BCL2 NM_000633:exon2:c.C176A-(p.P59Q) / /709 Heterozygous BCL2 NM_000633:exon2:C20T-(p.T7I) / /8032 Heterozygous EP300 NM_001429:exon27:c.4373_4375del-(p.1458_1459del) / /11990 Heterozygous MYD88 NM_002468:exon5:c.T778C-(p.L265P) / /13194 Heterozygous IRF4 NM_002460:exon2:c.G53A-(p.S18N) / /4374 Heterozygous PIM1 NM_002648:exon3:c.237delG-(p.E79fs) / /4030 Heterozygous 1768 M 83 IV ABC PIM1 NM_002648:exon4:c.G382C-(p.D128H) / /3812 Heterozygous PRDM1 NM_001198:exon2:c.G284A-(p.S95N) / /3724 Heterozygous 1.82 PRDM1 NM_001198:exon5:c.1103_1112del-(p.368_371del) / /11689 Heterozygous MYC NM_002467:exon2:c.C145T-(p.Q49X) / /979 Heterozygous CD79B NM_000626:exon5:c.A587C-(p.Y196S) / /9060 Heterozygous Supplementary Table 1: Clinical characteristics and list of somatic variants (insertion/deletion/ single nucleotide variant) detected by sequencing in tumor DNA and cell-free plasma circulating DNA. ABC: Activated B-Cell like; GCB: Germinal Center B-cell like; In/del: insertion/deletion; LDH: Lactate dehydrogenase; IPI: International Prognosis Index; SNV: single nucleotide variant; UPN: Unique Personal Number; ULN: upper limit value; VAF: variant allele frequency 6 4