Linfoma di Burkitt (L-B)

Linfoma di Burkitt
(Burkitt)
Aims
….Attitudes are more
important than abilities
Motives are more
important than methods
Character is more
important than cleverness
And heart takes precedence
over the head…..
Denis P. Burkitt, 1991
LINFOMA DI BURKITT (LBT)
endemico:
zone centrali dell’Africa (malaria)
età pediatrica; sedi: mandibola, ovaio
sporadico:
paesi occidentali
età variabile; sedi: intestino, gonadi,
SNC
HIV-correlato: SNC, etc.
Buona risposta in età pediatrica con terapie aggressive
FORMA ENDEMICA
(Lancet Oncology 2004; 5:738-46)
Patogenesi del linfoma di Burkitt endemico
legata alla cooperazione di:
- EBV (immortalizzazione),
- malaria (spinta proliferativa),
- Arbovirus trasmessi da mosche (stimolo
immunitario),
- Euphorbia tirucalli (danno al DNA),
- traslocazione coinvolgente c-MYC.
Hum. Pathol., 39:817-823, 2008.
HIV infection and c-MYC status in endemic Burkitt lymphoma
Campidelli, C., Gazzola, A., Vitone, F., Tumwine, L., and Pileri, S.A.
Hum. Pathol., 39:1408-9, 2008.
Patient and material collection
•
95 cases from Ugandan tribes
•
Most cases occurred in patients aged 9-10
•
Site of involvement:
- lymph node (34 cases)
- abdomen
(26 cases)
- gonads
(25 cases)
- jaw
(10 cases)
HIV-status
• None of the samples showed HIV integration
• HIV-1 DNA was investigated by:
RT-PCR amplifying gag region (142 bp)
nested PCR amplifying env region (248 bp)
Driver GA et al. J Virol Methods, 2007
Brachtel EF et al. AIDS Res Hum Retroviruses, 2002
Strappe PM et al. J Virol Methods, 1998
LINFOMA DI BURKITT
MORFOLOGIA:
endemico/sporadico= isomorfi
taglia media omogenea
cromatina reticolare
2-6 nucleoli
citoplasma basofilo/vacuolato
crescita coesiva
mitosi numerosissime
aspetto a cielo stellato
LINFOMA DI BURKITT
FENOTIPO:
SIgM+/marcatori B+
CD10+
Bcl-6+
Bcl-2c-myc+
Mib-1+ = 100%
(CD30; CD138: e/HIV+)
CD10
Bcl-6
Bcl-2
Ki-67
MIB1
BL subtypes are all related to GC cells & presents
several deregulated genes and cellular programs
1,000 genes
Naïve
Germinal center B -cells
Naive
Memory
Germinal center B cells
BL
Memory
BL
N/M
N/M
GC
GC
-1
+1
Grey zone
SPARC
• immune response
• proliferation (MYC)
• adhesion
• NOTCH signaling
BL
Germinal Center B cells
Early or re-entering germinal centre B-cells!
CD138
CD30
GENOTIPO
LBT endemico:
EBV+
t(8;14)
LBT sporadico :
EBV+: 25% HIV50% HIV+
t (2;8)
t(8;22)
diversi breakpoints
Sul cromosoma 8: gene c-MYC
EBER
GENOTIPO
LBT endemico:
EBV+
t(8;14)
LBT sporadico :
EBV+: 25% HIV50% HIV+
t (2;8)
t(8;22)
diversi breakpoints
Sul cromosoma 8: gene c-MYC
Question
• Are the three subtypes of Burkitt
lymphoma different diseases or different
features of the same disease?
Gene expression profiling of Burkitt
Lymphomas
SPORADIC!
Davè S et al, NEJM 2006
Hummel M et al, NEJM 2006
Piccaluga PP, De Falco G, Kustagi K,
Gazzola A, Astolfi A, Agostinelli C,
Leucci E, Onnis A, Tripodio C,
Sapienza MR, Bellan C, Lazzi S,
Tumwine L, Mawanda M, Ogwang M,
Calbi V, Formica S, Califano A, Pileri
SA and Leoncini L: Gene expression
analysis uncovers similarity and
differences among Burkitt lymphoma
subtypes. Blood 2011, 117:3596-3608.
Molecular profiling of
BL subtypes
BL is a unique molecular
entity
Burkitt lymphoma
Follicular lymphoma
Diffuse large B-cell lymphoma
Primary mediastinal B-cell lymphoma
Chronic lymphocytic leukemia
Normal B-cells
Though similar, BL subtypes present
differences in their GEPs
eBL
sBL
HIV-BL
eBL
eBL
HIV-BL
HIV -BL
sBL
sBL
Though similar, BL subtypes present
differences in their GEPs
eBL
HIV-BL
eBL and HIV-BL: 16 genes
(almost identical),
eBL
sBL
eBL & sBL: 254 genes, including
cell cycle regulation.
GSEA showed that eBL and sBL differ for the
expression of genes related to the RBL2 network
eBL
sBL
Accordingly, RBL2 malfunction is relevant for eBL but doesn’t affect sBL.
RBL2 functional network identified by
ARACNe
The results do not vary by
subtracting the MYC network
BL molecular profile depends also on EBV status
A
A molecular signature
discriminated BLs according to
the presence of EBV in both a
training (A) and a test (C) set of
cases (chi-sq., p<0.0001).
C
Such genes were significantly
related to apoptosis regulation
sBL &
HIV+
regulation of transcription from Pol II
promoter
B
sBL &
HIV+
D
induction of apoptosis by extracellular
signals
regulation of programmed cell death
positive regulation of programmed cell
death
induction of apoptosis
positive regualtion of apoptosis
induction of programmed cell death
development
male gamete generation
Apoptosis!
spermatogenesis
gametogenesis
20 viral miRNAs
MYC-translocation negative Burkitt lymphoma
differs from classical cases
A
B
B
A
Unsupervised
N=974 genes
Supervised
MYC-negative
C
1
0.5
BL MYC-neg
BL MYC-pos
0
Grey zone
-0.5
-1
MYC-positive
N=11,885 genes
Recurrently mutated genes were found in Burkitt
lymphoma by NGS
In 70% of sBL cases,
mutations affecting the transcription
factor TCF3 (E2A) or its negative
regulator ID3 fostered TCF3 dependency.
TCF3 activated the pro-survival PI(3)
kinase pathway in BL, in part by
augmenting constitutive B cell receptor
signaling.
Schmitz R et al, Nature 2012
Richter J et al, Nature Genetics 2012
High throughput sequencing to
further explore these issues
TP53
CCND3
ID3
N=26
(4.0%)
MYC
DDX3X*
GNA13
TCF3
SMARCA4
PDCD11
NCOR2
Burkitt Lymphoma RNA Sequencing
ILLUMINA HiScanSQ
Bologna University, Columbia University and Siena University
21 Endemic Burkitt Lymphoma Samples Sequencing Stats
• 2x75 bp Paired End Reads
• Total # of reads: 1,755 millions
(average 83.5 millions for sample)
cases
were pretty well preserved
• Total # of African
bases: 131
Gigabases
in RNAlater
(average 6 Gigabases for sample)
• Average theoretical coverage
54X
• 80% base>Q30, mean quality
score 32,55