Linfoma di Burkitt

(Burkitt)

Aims….Attitudes are more

important than abilitiesMotives are more

important than methodsCharacter is more

important than clevernessAnd 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 occidentalietà 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.

HIV infection and c-MYC status in endemic Burkitt lymphomaCampidelli, C., Gazzola, A., Vitone, F., Tumwine, L., and Pileri, S.A.Hum. Pathol., 39:1408-9, 2008.

Hum. Pathol., 39:817-823, 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, 2007Brachtel EF et al. AIDS Res Hum Retroviruses, 2002 Strappe PM et al. J Virol Methods, 1998

LINFOMA DI BURKITT

MORFOLOGIA: endemico/sporadico= isomorfitaglia media omogeneacromatina reticolare2-6 nucleolicitoplasma basofilo/vacuolatocrescita coesivamitosi numerosissimeaspetto a cielo stellato

LINFOMA DI BURKITT

FENOTIPO: SIgM+/marcatori B+

CD10+

Bcl-6+

Bcl-2-

c-myc+

Mib-1+ = 100%(CD30; CD138: e/HIV+)

CD10 Bcl-6

Bcl-2

Ki-67MIB1

BL subtypes are all related to GC cells & presents several deregulated genes and cellular programs

SPARC BLGerminal Center B cells

+1 -1

Naïve

Germinal center B -cells

Memory

BL

Grey zone

GC N/M

+1 -1

NaiveGerminal center B cellsMemoryBL

Grey zone

GC N/M

1,000 genes

• immune response • proliferation (MYC)• adhesion • NOTCH signaling

Early or re-entering germinal centre B-cells!

CD138

CD30

GENOTIPO

LBT endemico: EBV+

t(8;14)

LBT sporadico : EBV+: 25% HIV-

50% 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% HIV-

50% 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

Davè S et al, NEJM 2006Hummel M et al, NEJM 2006

SPORADIC!

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.

Burkitt lymphomaFollicular lymphomaDiffuse large B-cell lymphomaPrimary mediastinal B-cell lymphomaChronic lymphocytic leukemiaNormal B-cells

Molecular profiling of BL subtypes

BL is a unique molecular entity

Though similar, BL subtypes present differences in their GEPs

eBL sBL HIV-BLeBL sBL HIV-BL eBLHIV-BLsBL

eBLHIV -BLsBL

eBLsBL

eBLHIV-BL

eBL and HIV-BL: 16 genes (almost identical),

eBL & sBL: 254 genes, including cell cycle regulation.

Though similar, BL subtypes present differences in their GEPs

GSEA showed that eBL and sBL differ for the expression of genes related to the RBL2 network

RBL2 functional network identified byARACNe

eBLsBL

The results do not vary bysubtracting the MYC network

Accordingly, RBL2 malfunction is relevant for eBL but doesn’t affect sBL.

BL molecular profile depends also on EBV status

Dregulation of transcription from Pol IIpromoter

induction of apoptosis by extracellularsignals

regulation of programmed cell death

positive regulation of programmed celldeath

induction of apoptosis

positive regualtion of apoptosis

induction of programmed cell death

development

male gamete generation

spermatogenesis

gametogenesis

B

Apoptosis!

AA CCA 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).

Such genes were significantly related to apoptosis regulation

20 viral miRNAs

sBL &HIV+

sBL &HIV+

MYC-translocation negative Burkitt lymphoma differs from classical cases

AA

B

N=11,885 genes

Unsupervised

N=974 genesN=974 genes

A BB

-1

-0.5

0

0.5

1

BL MYC-negBL MYC-pos

Grey zone

MYC-negative

MYC-positive

C

-1

-0.5

0

0.5

1

BL MYC-negBL MYC-posBL MYC-negBL MYC-pos

Grey zone

MYC-negative

MYC-positive

C

Supervised

Recurrently mutated genes were found in Burkitt lymphoma by NGS

Schmitz R et al, Nature 2012Richter J et al, Nature Genetics 2012

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.

High throughput sequencing to further explore these issues

N=26(4.0%)

ID3

MYC

DDX3X*

CCND3

NCOR2

TP53

GNA13TCF3

SMARCA4PDCD11

Burkitt Lymphoma RNA Sequencing

ILLUMINA HiScanSQ

Bologna University, Columbia University and Siena University

• 2x75 bp Paired End Reads

• Total # of reads: 1,755 millions (average 83.5 millions for sample)

• Total # of bases: 131 Gigabases(average 6 Gigabases for sample)

• Average theoretical coverage 54X

• 80% base>Q30, mean quality score 32,55

21 Endemic Burkitt Lymphoma Samples -Sequencing Stats

African cases were pretty well preservedin RNAlater