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LETTER TO THE EDITOR
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Analysis of mutational signatures in exomes from B-cell lymphoma cell lines suggest
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APOBEC3 family members to be involved in the pathogenesis of Primary Effusion
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Lymphoma
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R. Wagener1, L.B. Alexandrov2,3, M. Montesinos-Rogen4, M. Schlesner5, A. Haake1, H.G. Drexler6, J.
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Richter1, G.R. Bignell2, U. McDermott2, R. Siebert1
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Institute of Human Genetics, Christian-Albrechts-University Kiel & University Hospital Schleswig-
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Holstein, Campus Kiel, Kiel Germany
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Cancer Genome Project, Wellcome Trust Cancer Sanger Institute, Wellcome Trust Genome Campus,
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Hinxton, Cambridge, UK
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Theoretical Division, Los Alamos National Laboratory, Los Alamos, New Mexico, USA
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Institute for Neuropathology, University Hospital of Cologne, Cologne, Germany
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Division of Theoretical Bioinformatics, Deutsches Krebsforschungszentrum Heidelberg (DKFZ),
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Heidelberg, Germany
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Leibniz-Institute DSMZ- German Collection of Microorganisms and Cell Cultures, Braunschweig,
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Germany
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Supplement
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Deciphering mutational signatures
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Whole exome sequencing of 36 mature B-cell lymphoma cell lines, including 4 PEL cell lines (BC-1,
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BC-3, CRO-AP2, JSC-1), twelve Burkitt lymphoma cell lines (BL-41, CA46, DAUDI, DG-75, EB-2, EB-3,
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GA-10, JIYOYEP, NAMALWA,RAJI, RAMOS, ST486) and 25 other mature B-cell lymphoma cell lines
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(A4-FUKUDA, A3-KAWAKAMI, CTB-1, DB, DOHH-2 FARAGE, HT, KARPAS-1106P, KARPAS422, MC116,
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NU-DUL-1, OCI-LY19, RC-K8, RL, SC-1, SCC-3, SU-DHL-4, SU-DHL-5, SU-DHL-6, SU-DHL-8, SU-DHL-10,
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SU-DHL-16, TK, VAL, WSU-DLCL2) was performed as part of the Cell Lines Project at The Welcome
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Trust Sanger Centre.
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After sequencing samples were filtered to remove common germline mutations based on sequencing
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data of approximately 8,000 samples: 1000 genomes (released March 29th 2012) - variants with a
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frequency > 0.0014, ESP6500 (released June 20th 2012) - variants with a frequency >= 0.00025,
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dbSNP (Ensembl 58) - variants with minor allele frequency, and ~350 in-house normal samples -
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variants where a mutation is seen in more than 1 normal sample. For more details see
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http://cancer.sanger.ac.uk/cancergenome/projects/cell_lines/. The mutational data entering the
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present
COSMIC
Cell
Line
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(http://cancer.sanger.ac.uk/cancergenome/projects/cell_lines/).
The
subsequent
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signatures analysis is based on downloading VCF files from the website and analyzing all available
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mutations types (coding, noncoding, synonymous, non-synonymous, etc.).
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The assignment of mutational signatures bases on the 27 recently described distinct consensus
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mutational signatures.1 All possible combinations of at least seven of these mutational signatures
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were evaluated for each cell line by minimizing the constrained linear function:
analysis
are
available
through
the
project
database
mutational
𝑁
min
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𝐸𝑥𝑝𝑜𝑠𝑢𝑟𝑒𝑠𝑖 ≥0
⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗ − ∑(𝑆𝑖𝑔𝑛𝑎𝑡𝑢𝑟𝑒
⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗𝑖 ∗ 𝐸𝑥𝑝𝑜𝑠𝑢𝑟𝑒𝑖 )||
||𝐶𝑒𝑙𝑙𝐿𝑖𝑛𝑒
𝑖=1
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Here, ⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗
𝐶𝑒𝑙𝑙𝐿𝑖𝑛𝑒 and ⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗
𝑆𝑖𝑔𝑛𝑎𝑡𝑢𝑟𝑒𝑖 represent vectors with 96 components (corresponding to the six
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somatic substitutions and their immediate sequencing context) and 𝐸𝑥𝑝𝑜𝑠𝑢𝑟𝑒𝑖 is a nonnegative
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natural scalar, 𝐸𝑥𝑝𝑜𝑠𝑢𝑟𝑒𝑖 ∈ 𝒩0 , reflecting the number of mutations contributed by this signature. 𝑁
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reflects the number of signatures found in a cell line and all possible combinations of consensus
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mutational signatures for N between 1 and 7 were examined for each cell line. This resulted in
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1,285,623 solutions per sample and a model selection framework based on Akaike information
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criterion was applied to these solutions to select the optimal decomposition of mutational
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signatures.
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qPCR based expression analyses
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For the expression analyses, the twelve Burkitt lymphoma cell lines BL-2, BL-41, BL-70, BLUE-1, CA46,
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DG-75, RAMOS, U-698-M, DAUDI, EB-1, NAMALWA, RAJI, the ten diffuse large B-cell lymphoma cell
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lines HT, KARPAS422, OCI-LY7, RC-K8, RIVA, SU-DHL-5, SU-DHL-6, SU-DHL-8, SU-DHL-10, U2932 have
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been obtained from the German Collection of Microorganisms and Cell Cultures GmbH
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(Braunschweig, Germany). Cell pellets from the nine primary effusion lymphoma cell lines BC-1, BC-2,
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BC-3, BCBL-1, BCP-1, CRO-AP2, CRO-AP3, CRO-AP5, CRO-AP6 were kindly provided by Hans Drexler,
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refer to 2 for an overview of the cell line origins. Cells were grown in RPMI medium supplemented
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with 10% FBS and 1% Glutamine. The identity of all cell lines has been verified using the STEM ELITE
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ID Kit from Promega (Mannheim, Germany).
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RNA extraction was performed using the RNeasy Mini Kit from Qiagen (Hilden, Germany) according
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to manufacturers. For quantitative PCR (qPCR) analysis of gene expression, total RNA was reverse
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transcribed using the QuantiTect Reverse Transcritption Kit from Qiagen using 1 µg of RNA. qPCR was
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performed on a Roche Lightcycler 480 instrument using the QuantiTect SYBR Green PCR Kit (Qiagen).
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Gene expression was normalized to that of the housekeeping genes GUSB and HPRT1 using
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predesigned QuantiTect Primer Assays from Qiagen. The APOBEC3B primers were as follows: 5’-
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GCACCGCACGCTAAAGGAG-3’ and 5’-CCACCTCATAGCACAAGTAGG-3’. APOBEC3C primers were used
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as published by Burns et al.3
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Table SI: Overview of PEL cell lines and karyotypes
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Cell line information was downloaded from the homepage of the German Collection of
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Microorganisms and Cell Cultures (DSMZ) (http://www.dsmz.de/home.html). Sole exceptions are
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BCP-14 and JSC-15.
Cell line
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Diagnosis
Age
Gender
viral status
of cell line
BC-1
PEL + AIDS
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m
HHV8+,
EBV+
BC-2
PEL + AIDS
31
m
HHV8+,
EBV+
BC-3
PEL
85
m
HHV8+
BCBL-1
Kaposi`sarcoma,
PEL+AIDS
40
m
HHV8+
BCP-1
Kaposi`sarcoma,
PEL
94
m
HHV8+,
EBV+
CRO-AP2
Kaposi`sarcoma,
PEL+AIDS
49
m
HHV8+,
EBV+
CRO-AP3
PEL + AIDS
42
m
HHV8+
CRO-AP5
Kaposi`sarcoma,
PEL+AIDS
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m
HHV8+,
EBV+
Karyotype
50(47-51)<2n>XY, +7, +8, +8, +mar,
der(3)ins(3;1)(q28;q11q42), inv(8)(p23q24)x2,
der(9)t(2;9)(q32;q35), del(12)(q24),
der(15)t(4;15)(q31;p13), der(16)t(15;16)(p13;q15)
97-99<4n>XXYY,-2,+10,+12,+12,-14,-14,-17,18,+19, +19,-21,+8,-10
mar,add(X)(q24)x2,del(7)(q21.3 )x2, add(11)(q22),
add(11)(q24) , add(12)(q13), add(12)(q24)x1-2,
der(14)t(7;14)(q21;p11),
der(14)t(14;2)(q32;q21)x2-4, add(16)(q21)x1-2
47(43-48)<2n>X, -Y, +7, +8, der(1)t(1;16)(q44;q13),
del(7)(p21.1p21.2), t(8;15)(p21;q26), del(13)(q13),
der(14)t(12;14)(q11;p11),t(16;22)(q13;q12),
der(19)t(19;19)(p13.1;q13.3), del(20)(p11.2p11.3)
91-100<4n>XX, -Y, -Y, +2, +2, +6, +7, +7, +8, -9, -10,
-11, +12, -13, -13, +15, -16, -22, -22, +2, -5mar,
dup(1)(q12q44), del(1)(p11)dup(1)(q12q44),
del(2)(p11)x1-2, der(2)t(2;5)(q35;?q32Gende)
t(5;8)(?q35;q22), der(4)t(4;8)(q24;q24),
t(6;17)(p23;q21), del(9)(q12q21),
der(11)t(11;15)(q23;q21),
der(11)t(11;15)(q25;q25),
der(13)t(13;13)(p12;q21)x1-2,
der(14)t(4;14)(q31;q32)x2,
der(14)t(14;22)(p12;q12)t(4;14)(q31;q22)x2,
der(15)t(8;15)(q21;q25)
der(X)t(X;12;14)(q12;q11:q22-23;q23-24)
48(47-48)<2n>X, +X, -Y, +12, add(?Y)(p11),
add(1)(q43.1), der(15)t(8;15)(q11;q25),
der(22)t(1;22)(q21;q13) - sideline with add(9)(q13)
80-88<4n>XX, -Y, -Y, -1, -2, +6, -7, -10, -13, -13, -22,
-22, +6mar, i(1q), add(2)(p2?), add(3)(q2?7),
der(4;9)(q10;q10)x1-2, del(5)(p13p15)x1-2,
del(5)(q2?1)x1-2,
der(6)t(6;7)(q15;q11)add(7)(q3?5), del(8)(p11),
del(9)(p21), add(9)(p11), del(11)(q2?2q2?3)x2,
der(12)t(2;12)(p12;q24.32), add(13)(p11),
t(14;19)(q11;q13)x2,
der(15)t(1;?)(?15)(q21;)(?;q11), ider(?)t(8;?)(q11;?)
44-49<2n>X, -Y, +8, +2mar, del(4)(q25),
add(8)(p11), dup(12)(q11q14), del(14)(q24)
CRO-AP6
PEL + AIDS
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m
HHV8+
48(45-48)<2n>XY, +16, +mar,
der(11)t(4;11)(q28;q24),
der(13)dup(13)(q1?q3?)t(13;14)(q3?;q21),
der(14)t(13;14)(q3?;q21), add(16)(q24)(hsr),
i(21)(q10); carries hsr on der(13) and der(16)
JSC-1
PEL
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m
HHV8+,
EBV+
45,XY,der(1)t(1;8)(q42;q11),add(6)(q25),10,add(14)(q24).
Gender: M denotes male and f denotes female, n.d., no data available.
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References
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1 Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SAJR, Behjati S, Biankin AV et al. Signatures of
mutational processes in human cancer. Nature 2013; 500: 415–421.
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2 Drexler HG, MacLeod RAF, Nagel S, Dirks WG, Uphoff CC, Steube KG et al. Guide to LeukemiaLymphoma Cell Lines on CD. ASH Annu Meet Abstr 2005; 106: 4340.
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3 Burns MB, Lackey L, Carpenter MA, Rathore A, Land AM, Leonard B et al. APOBEC3B is an
enzymatic source of mutation in breast cancer. Nature 2013; 494: 366–370.
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4 Boshoff C, Gao SJ, Healy LE, Matthews S, Thomas AJ, Coignet L et al. Establishing a KSHV+ cell line
(BCP-1) from peripheral blood and characterizing its growth in Nod/SCID mice. Blood 1998; 91:
1671–1679.
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5 Cannon JS, Ciufo D, Hawkins AL, Griffin CA, Borowitz MJ, Hayward GS et al. A new primary effusion
lymphoma-derived cell line yields a highly infectious Kaposi’s sarcoma herpesvirus-containing
supernatant. J Virol 2000; 74: 10187–10193.
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