96. Jahrestagung der Deutschen Gesellschaft für Pathologie e. V ...

Abstracts
Methods. Forty-three patients from 3 different Swiss hospitals were included
in the study. The tumors were diagnosed between 1972 and 2009.
The protein expression profile was assessed by immunohistochemistry.
Results. 39 of the tumors showed centroblastic and 1 immunoblastic morphology,
three were not classifiable. All cases were positive for CD79a,
followed by CD20 and PAX5 (95% of cases) and CD19 (93%). Most cases
(68%) expressed the post-germinal center (GC) marker FOXP1 and
21% expressed MUM1, while the GC markers CD10, LMO2, BCL6 and
GCET1 were expressed in 27, 16, 8 and 6%, respectively (cut-off levels for
the respective markers were determined by ROC). BCL2 was expressed
on >50% of the tumor cells in 69% of cases. 83% of cases were phenotypically
classifiable as non-GC DLBCL according to the Tally algorithm.
There was no evidence for EBV- or HHV8-association. 70% of the tumors
showed active STAT signaling by expression of either pSTAT1 or pSTAT3,
but not pSTAT5. p53 was expressed in 12% of cases, but p21 staining (p21/
p53) did not suggest presence of TP53 mutations.. Mean mitotic index
was 18/mm2, median MIB1 labeling index was 40% (±25%). Tumors with
lymphoepithelial lesions in seminiferious tubules showed a lower mitotic
activity, although the association was weak. Interestingly, one tumor was
positive for OCT4. All 43 cases were negative for NUT1 and PLAP. Only
limited clinical data were available: mean age at diagnosis was 69 years
(range: 43–87 years, n=41). There was no side predilection of the tumors.
One tumor was bilateral at diagnosis, one tumor presented simultaneously
in the testis and the CNS. Of ten tumors, five did not relapse
(mean follow up time 48 months). Five tDLBCL relapsed, thereof two in
the contralateral testis, two in the CNS and one in the skin.
Conclusions. We conclude that tDLBCL are predominantly centroblastic
and of non-GC phenotype. Since occasionally tDLBCL can express germ
cell markers or be CD20-negative, multimarker phenotyping is important
for lineage determination. There was no shift of morphology or protein
expression profile over time. tDLBCL have active STAT signalling
mediated through pSTAT1 and pSTAT3. tDLBCL are of non-/post-GC
origin and not hyper-proliferative. TP53 mutations are unlikely.
DO-060
C-MYC aberrations characterize a subset of patients with diffuse
large B-cell lymphoma with poor outcome when treated with
rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone
A . Tzankov1 , M . Gerhard1 , S . Dirnhofer1 , C . Visco2 , K . Young3 1 2 University Hospital Basel, Pathology, Basel, Switzerland, San Bortolo Hospital,
Internal Medicine, Vicenza, Italy, 3The University of Texas MD Anderson
Cancer Center, Pathology, Houston, United States
Aims. Diffuse large-B cell lymphoma (DLBCL) exhibits variable outcomes
and risk assessment is based on the international prognostic index
(IPI), which takes into account primarily patient-related parameters.
Gene expression profiling (GEP) can stratify patients with different
prognoses into germinal center B-cell (GCB) and activated B-cell subtype
(ABC). These groups remain of prognostic importance with the addition
of rituximab (R) to chemotherapy. The prognostic role of C-MYC
gene status in the era of modern treatment is still debatable.
Methods. To address this question, we analyzed C-MYC gene abnormalities
by interphase fluorescence in situ hybridization (FISH) utilizing
break-apart- (BAP) and a IgH/C-MYC double-fusion (DFP) probes in
601 patients with de novo DLBCL treated with R, cyclophosphamide,
doxorubicin, vincristine, prednisone (R-CHOP) and 332 patients treated
with CHOP; all patients had clinical follow-up and GEP data.
Results. C-MYC gene abnormalities were detected in 67 of 672 evaluable
cases (10%), including 7 amplifications (2 ABC, 5 GCB), 4 rearrangements,
detectable only with the DFP (2 ABC, 2 GCB), 23 rearrangements,
detectable only with the BAP (5 ABC, 18 GCB) and 33 rearrangements,
detectable with both the BAP and DFP (12 ABC, 21 GCB; p=0.032 for
the differences between ABC and GCB). In multivariable models the last
two C-MYC aberration types represented IPI- and GEP-independent
30 | Der Pathologe · Supplement 1 · 2012
prognostic factors for event-free survival in R-CHOP treated patients
(p=0.003; relative risk 1.46) and in patients with GCB (p=0.014; relative
risk 1.29), but (though being of prognostic significance in univariable
models) not in CHOP treated ones and in ABC, where IPI was the sole
prognosticator.
Conclusions. C-MYC aberrations are observed in 10% of DLBCL, more
commonly in GCB cases. Alternative C-MYC rearrangements with non-
IgH partners, which are detectable only with BAP, account for a third of
all C-MYC structural genetic abnormalities. C-MYC aberrations detected
by BAP add IPI independent prognostic information for individual
DLBCL risk estimation in R-CHOP treated cases.
DO-061
MYC-binding sites in Burkitt lymphoma identified by deep
sequencing
V . Seitz1 , P . Butzhammer2 , B . Hirsch1 , J . Hecht3 , I . Gütgemann4 , A . Ehlers1 ,
D . Lenze1 , E . Oker1 , A . Sommerfeld1 , E . von der Wall1 , C . König5 , C . Zinser6 ,
R . Spang2 , M . Hummel1 1 2 Institute of Pathology, Charité – University Medicine, Berlin, University
of Regensburg, Institute for Functional Genomics, Regensburg, 3Charité –
University Medicine, Berlin-Brandenburg Center for Regenerative Therapies
(BCRT), Berlin, 4University Hospital of Bonn, Department of Pathology, Bonn,
5 6 imaGenes GmbH, Source BioScience, Berlin, Genomatix Software GmbH,
Genomatix Personalized Medicine, München
Aims. MYC is a key transcription factor involved in central cellular processes
such as regulation of the cell cycle, histone acetylation and ribosomal
biogenesis. It is overexpressed in the majority of human tumors
including aggressive B-cell lymphoma. Especially Burkitt lymphoma
(BL) is a highlight example for MYC overexpression due to a chromosomal
translocation involving the c-MYC gene. However, so far genomewide
analysis of MYC-binding sites by chromatin immunoprecipitation
(ChIP) followed by next generation sequencing (ChIP-Seq) has not been
conducted in BL. Therefore, our objective was the precise and representative
consideration of the MYC landscape in BL due to a much higher
coverage of MYC-binding sites employing ChIP-Seq.
Methods. We carried out ChIP in 5 human BL cell lines, employing a
MYC-specific antibody followed by next generation sequencing of the
precipitated DNA fragments. To assess the functional consequences of
MYC binding, the ChIP-Seq data were supplemented with siRNA-mediated
knock-downs of MYC in BL cell lines followed by gene expression
profiling.
Results. Our ChIP-Seq analysis gave rise to 7,054 MYC-binding sites after
bioinformatics analysis of a total of approx. 19 million sequence reads. In
line with previous findings, binding sites accumulate in genes known to
be involved in the cell cycle, ribosomal biogenesis, histone acetylation
and DNA-methylation demonstrating a regulatory role of MYC in these
processes. Unexpectedly, MYC-binding sites also accumulate in many
B-cell relevant genes and expression analysis after knock-down of MYC
by siRNA identified genes involved in the B-cell function which are upregulated
in response to MYC silencing.
Conclusions. The 7,054 MYC-binding sites identified by our ChIP-Seq approach
greatly extend the knowledge regarding MYC binding in BL and
shed further light on the enormous complexity of the MYC regulatory
network. Especially our observations that (i) many B-cell relevant genes
are targeted by MYC and (ii) that MYC down-regulation leads to an upregulation
of B-cell genes highlight an interesting aspect of BL biology.