Supplementary Material and Methods
Patients and samples
To uncover the mutational spectrum of germinal center-derived aggressive B-NHL
including BL, BCL-U and GCB DLBCL, a search in the archives of the Institute of Pathology,
University of Würzburg (Würzburg, Germany) and the Department of Clinical Pathology,
Robert-Bosch-Krankenhaus (Stuttgart, Germany) was conducted. Ethics approval for this
study was granted by the Local Ethics Committees. We selected formalin fixed and paraffin
embedded (FFPE) tumor tissues of 108 aggressive B-NHL based on well preserved
morphological features of the cases and the availability of sufficient material to conduct the
analyses with good DNA quality (see below for DNA quality check). Samples obtained
between the years 1993 and 2013 were included and all cases represented tumor specimens
at primary diagnosis. Standard immunohistochemical markers including at least CD20,
CD10, BCL2, BCL6 and Ki-67 were available in all cases. A re-review of all cases by expert
hematopathologists (A.R., G.O.) resulted in the definition of 31 BL, 24 BCL-U cases and 53
DLBCL cases, according to the 2008 WHO classification.1 All cases were EBV-negative.
Specifically, BCL-U cases showed a morphology intermediate between BL and DLBCL with
cytological features that were not acceptable for the diagnosis of BL. Alternatively, the
immunophenotype was not consistent with the diagnosis of BL (e.g. strong BCL2 expression
that occurred in ~30% of these cases). Almost all BCL-U cases (23/24) carried a MYC
translocation, and many of them an additional BCL2 and/or BCL6 translocation (see below).
DLBCL cases all belonged to the GCB DLBCL subgroup according to the Hans criteria.2
Cytologically, most of these DLBCL were of centroblastic or centroblastic polymorphic
morphology. DLBCL cases were enriched for tumors with MYC translocation (see below).
Fluorescence in situ hybridization (FISH)
Interphase FISH to detect rearrangements of the MYC-, BCL2- and BCL6-loci was
performed on standard whole sections of FFPE tissues using break-apart probes for the
respective loci (Vysis LSI® MYC-, Vysis LSI® BCL2- and Vysis LSI® BCL6-dual color
break-apart rearrangement probes, Vysis/Abbott Molecular Diagnostics, WiesbadenDelkenheim, Germany). The cutoff levels were 15% (MYC), 12% (BCL2), and 13% (BCL6),
as defined in our standard operating procedures. The evaluation of the signal distributions
was carried out by two independent experienced technicians. As a general approach, we first
performed FISH for the MYC locus (if it had not been done before as part of the routine
diagnostic work-up, e.g. in Burkitt lymphomas), and if a break in the MYC locus was
observed, FISH for the detection both BCL2 and BCL6 breaks was additionally performed.
Using this strategy, we were able to subcategorize the 53 DLBCL cases into three
groups, namely 28 DLBCL cases without MYC translocation (DL (GCB)), 16 DLBCL cases
with a MYC-only translocation (DL-MYC) and 9 DLBCL with MYC translocation plus
additional breaks in the BCL2 and/or BCL6 loci (‘double hit’/’triple hit’ DLBCL, DL-DH/TH). In
summary, the following five subgroups of germinal center-derived aggressive B-NHL were
analyzed: BL, BCL-U, DL-DH/TH, DL-MYC and DL (GCB). The basic patient characteristics
are summarized in supplementary Table 3. Representative morphological features for each
of the five groups are displayed in supplementary Figure 1.
DNA isolation and DNA quality check
Serial 3-μm sections were cut from FFPE specimens and the first section was stained
with hematoxylin-eosin (HE) to evaluate the tumor cell content. If the tumor cell content was
less than 80%, macrodissection of tumor cell-rich areas (excluding normal adjacent tissue,
capsular tissue or perinodal soft tissue) was performed. No corresponding germline DNA
was available from the patients. After deparaffinization and hydration, DNA was isolated with
the QIAmp DNA mini kit (QIAGEN GmbH, Hilden, Germany). To check for DNA quality, we
performed a conventional PCR by amplifying ACTB (NM_001101) and GAPDH
(NM_002046), with product sizes of 254bp and 263bp, respectively. The primers for ACTB
and GAPDH are described in supplementary Table 4. The PCR condition was the same
compared to the amplification procedure on the 48.48 Fluidigm Access Array™ System
(Fluidigm Europe B.V.). When the intensity of the product for both ACTB and GAPDH was
weak, a given sample was excluded from our cohort.
Mutation analysis
Regions of interest from ten genes were selected for mutational analysis according to
previous reports demonstrating high mutation rates in BL or GCB DLBCL. These are
summarized in Supplemental Table 5 and include ID3 (NM_002167.4), TCF3
(NM_001136139.2), CCND3 (NM_001760.4), MYC (NM_002467.4), BCL2 (NM_000633.2),
EZH2 (NM_001203247.1), MEF2B (NM_001145785.1), CREBBP1 (NM_004380.2), EP300
(NM_001429.3) and SGK1 (NM_001143676.1). All reference sequences of targeted
amplicons were extracted from GRCh37. Primers for the respective amplicons were
designed by Primer3 (for detailed primer sequences see Supplementary Table 4). The length
of amplicons was between 220 and 270bp (average: 247bp). Before PCR reaction on the
Fluidigm Access Array™ IFC (Fluidigm Europe B.V., Amsterdam, Netherlands), we
preamplified templates so that amplicons of each sample could be amplified effectively.
Preamplification was accomplished in two multiplex-PCR reactions with primer pools of 23
and 24 primer sets, respectively, with final concentrations of 50 nM of each forward and
reverse target-specific primer (Supplementary Table 4). Fifty microgram of template DNA
and the primer pool described above was added to 10μl reaction volumes with mastermix
containing 0.5 U FastStart High Fidelity Enzyme Blend (Roche Diagnostics, Mannheim,
Germany), 1× FastStart High Fidelity Enzyme Buffer, 200μM each of dNTPs, 4.5mM MgCl2
and 5% DMSO. Preamplification reaction PCR cycles were as follows: 95°C 10 min, 15
cycles of 95°C for 15 sec, 60°C for 30 sec and 72°C for 1 min, and final elongation of 72°C
for 7 min. Following preamplification, Exo-SAP-It (Affymetrix ltd., High Wycombe, UK) was
added to each sample. Samples were then incubated at 37°C for 30 min and inactivated at
80°C for 15 min, followed by 4-fold dilution in PCR-grade water prior to amplification on the
Fluidigm Access Array™ IFC (Fluidigm Europe B.V., Amsterdam, Netherlands). Each
amplicon was then processed on the 48.48 Fluidigm Access Array™ System. Following the
collection of these libraries, each library was attached with sample specific barcodes. Finally,
all libraries were cleaned up twice using SPRI Ampure-XP DNA-binding paramagnetic beads
with a ratio of 1:1.3 (Beckman Coulter, Krefeld Germany) and quantified with the PicoGreenH
assay (Invitrogen, Darmstadt, Germany). The enriched pool of beads was used for massively
parallel sequencing in a Titanium PicoTiterPlateTM with Titanium reagents (Roche
Diagnostics) on the GS Junior instrument. Data analysis was accomplished by the GS
Amplicon Variant Analyzer (AVA) software version 2.5.3 (Roche Diagnostics) and all
variations were aligned to the reference sequence and annotated using SeattleSeq
Annotation 138. Synonymous variants and single nucleotide polymorphisms reported in the
database (dbSNP_138) with a population allele frequency above 1% were excluded from
further analysis. The average coverage per sample was ~177-fold. To screen for potential
false negatives in our variant calling approach, we performed Sanger sequencing for TCF3 in
all BL samples and EZH2 in all DLBCL samples. By this approach, we did not detect any
false negatives by Sanger validation. Variant calling yielded 653 variants <20% and 561
variants <10% of VAF. We performed Sanger validation for randomly selected variants that
had VAFs between 10-20% (n=42) and 5-10% (n=30), however we could not detect any
mutations in these cases by Sanger sequencing. Finally we set the cut-off threshold for
validation as VAF >20%. Importantly, all mutations that had a VAF >20% were confirmed by
Sanger sequencing (see Table S6). If the number of reads of an amplicon in a given sample
was below 30, Sanger sequencing was also performed to detect or exclude a variant. For
Sanger sequencing, PCR products were amplified by the same primer sets used in the
Fluidigm library preparation and amplicons were checked by standard agarose gel
electrophoresis and purified by Exo-SAP-IT. The PCR products were sequenced with the Big
Dye Terminator cycle sequencing kit (Life Technologies, Darmstadt, Germany) using the
3130xl Genetic Analyzer (Life Technologies).
Statistics
We performed statistical analysis using the SPSS software (IBM SPSS Statistics,
version 21). The Chi-square test or Fisher´s exact test was applied for categorical variables.
P values <0.05 were considered statistically significant.
Supplementary figures legends
Supplementary Figure 1: Representative morphology of the five aggressive, germinal
center-derived lymphoma subgroups investigated. (A) Burkitt lymphoma (case 6). (B) BCL-U
(case 34; tumor cells showed strong BCL2 expression). (C) DLBCL with ‘triple hit’
(MYC/BCL2/BCL6 translocation, case 38). (D) DLBCL with MYC-only translocation (case
49). (E) DLBCL (GCB-type), without MYC translocation (case 67).
Supplementary Figure 2: Mutation detection by Roche Junior and Sanger sequencing. (A)
Monoallelic mutation in ID3 (c.C190T, p.L64F), case 19. (B) Biallelic mutation of ID3, as
Roche Junior data shows c.C190T (p.L64F) and c.C166T (p.P56S) mutations in different
reads. (C) Biallelic mutation of ID3, as Roche Junior data shows c.C190del (p.L64fs) and
c.C157T (p.E53*) mutations in different reads.
Supplementary Figure 3: ID3 mutation status in each of the subgroups.
Reference
1.
2.
Swerdlow SH, Campo, E., Harris, N.L., Jaffe, E.S., Pileri, S.A., Stein, H., Thiele,
J., Vardiman, J.W (ed). WHO Classification of Tumours of Haematopoietic and
Lymphoid Tissues. IARC: Lyon, France, 2008.
Hans CP, Weisenburger DD, Greiner TC, Gascoyne RD, Delabie J, Ott G, et al.
Confirmation of the molecular classification of diffuse large B-cell lymphoma by
immunohistochemistry using a tissue microarray. Blood 2004 Jan 1; 103(1): 275282.