EBV AND MYC IN BURKITT LYMPHOMA: WHICH

IS THE MAIN ACTOR?

Presented by Ochieng’ Noel Onyango

 BACKGROUND
▪ BL - a high-grade B-cell non-Hodgkin's common pediatric
disease lymphoma with high proliferative index

1. Endemic Burkitts lymphoma

•Equatorial Africa (Lymphoma belt)

•High incidence among children
•Peak Incidence, 4-7 yrs, Males : Females 2:1
•Sites: Extra nodal sites 20% involve jaws, other facial bones but
rare in Leukemia.
•Correlation with incidence & climatic factors, Malaria.
•Associated with EBV (100%) and Malaria which are Ubiquitous in
the lymphoma belt.

Swerdlow et al., 2008. WHO Classification of Tumours of Hematopoietic and Lymphoid Tissues,
Fourth Edition. IARC WHO C. WHO Classification of Tumours,.
Co-factors in endemic Burkitt’s Lymphoma

EBV
 2. Sporadic Burkitt’s Lymphoma

Tumor histologically identical to BL identified in other parts of

the world, including US and Europe.

≈ 30% of childhood lymphomas, 1-2% of all lymphomas.

Median age of ≈ 12 yrs, Male: Female ratio - 2-3:1.

most common in the GI, Ovaries, Breast (at pregnancy, puberty).

EBV 20-30%

3. Immunodeficiency Associated BL

Presents early in the course of HIV with normal CD4 counts

Swerdlow et al., 2008. WHO Classification of Tumours of Hematopoietic and Lymphoid

Tissues, Fourth Edition. IARC WHO C. WHO Classification of Tumours,.
 Epstein-Barr Virus

EBV is a gamma herpesvirus linked to a number of lymphoid and epithelial

malignancies, including BL where its frequency ranges from 30% in sporadic
cases to 100% in the endemic ones.
The role of EBV to B-cell lymphomas pathogenesis is largely unknown.

✔ Viral genes are essential for initiating

lymphogenesis.
✔ Progressively, viral genome is lost after
initial infection and leaves mutations to
accumulate in the oncogenic cell.

The main problem with the hit-and-run

hypothesis is the lack of evidence of EBV in the
primary tumor.
 Role Of c-Myc Translocations In BL Development
The hallmark of all BL tumours is the translocation between the
c-MYC gene and (Ig) heavy or light chain loci. (MYC family include N &
L) ▪ MYC ≈ 100%
▪ Translocations t(8;14) or t(8;22), t(2;8)
▪ Rare MYC translocation negative cases????

80% - t(8;14) transl occurs in

IGH gene.
20% of cases are split between
the translocations with the
IGK and λGL (t(2;8) and
t(8;22) respectively).

Hecht BJL & Aster JC (2000). Molecular Biology of BL. Soc, 18(21), 3707–3721.
 c-MYC/MYC

The MYC transcription factor is pathologically activated in many human

malignancies: Genomic aberrations, Chrom ampln’s and Mutual
translocations.
However, genetic translocation is not the only means of MYC
deregulation. Other mutations of cell instability have been reported.
 Previous Studies

UNVEILING ANOTHER MISSING PIECE IN

EBV-DRIVEN LYMPHOMAGENESIS: EBV-ENCODED
MICRORNAS EXPRESSION IN EBER-NEGATIVE (BL)
CASES

(EBV LEAVES ITS MARK: NEW EVIDENCE OF HYPOTHESIS

IN B-CELL LYMPHOMA FROM NON-CONVECTIONAL
METHODS)

MYCN EXPRESSION DEFINES A NOVEL RARE SUBSET OF

BURKITT LYMPHOMA.
EBV LEAVES ITS MARK: NEW EVIDENCE OF HYPOTHESIS IN
B-CELL LYMPHOMA FROM NON-CONVENTIONAL METHODS

EBV might be associated with all BL cases, including those diagnosed as EBV
negative by routine methods IHC and EBV-encoded RNAs (EBER) in situ
hybridization–ISH] thanks to a mechanism of hit-and-run.

Challenge!!
These routine methods have a low specificity and accuracy.
 AIM #1
To identify the presence of EBV in a series of “EBV-Negative”
B-cell lymphomas by applying conventional and
non-conventional (IHC and ISH vs EBV viral load measurement; EBV-encoded miRNAs
detection, RNAscope assay)

METHOD
1) IHC scored as +ve or –ve for EBNA-1
10 BL,34 DLBCL, 50 FL, 44 cHL, 10 T-LL, 20 HCL, 5 MCL
In situ hybridation for EBER carried out in each sample.(EBER-ISH in all samples
)
2) We screened by RT-qPCR targeting regions of EBV genome: BamH1 W and EBNA-1.
3) In addition, we performed the RNAscope assay for targeting EBNA1 mRNA
molecule and identify which cells were EBV positive.
Mundo L. et al, Frontiers in Microbiology 2017
Mundo L et al, Hematological Oncology 2019
Results
Cases that were negative by in situ hybridization were
observed to be EBV‐positive by qPCR.
EBER-Ve-/qPCR Ve+ BL DLBL HL FL
A B C D

RNA Scope

E F G H
EBER-Ve+

ISH

I J K L
EBER-Ve-

ISH

We diagnosed 10 EBER-Ve+ and 6 EBER-Ve- BL, 4 EBER-Ve+ and 30 EBER-Ve- DLBCL, 47

EBER-Ve- and 3 EBER-Ve+ FL, 12 EBER-Ve+ and 32 EBER-Ve- HL.
 Case 1 BL

PCR FR2VHMIX-JH PCR FR2VHMIX-JH

260 bp
260 bp

-VE ISH +VE ISH

 Detection of EBV genome by reverse transcription assay.

A) EBNA-1(B) and (B). BamH1 W conserved region

The presence of the virus was also assessed and confirmed by the expression of
EBV‐encoded miRNAs (BART9‐5p, BART10‐3p, BART19‐3p).

Higher viral loads in BL‐EBER negative compared with the DLBCL, FL, HL
EBER‐Ve cases was detected.
Conclusion

EBV negative cases as defined by EBER status is in accord with the

hit and run hypothesis that claims the possibility that EBV genome
can be lost from human cells in each cell cycle.

Our findings highlight for the first time the possibility that EBV
might contribute to the development of more lymphomas than simply
those remaining viral genome‐positive.
 Study Number Two

MYCN EXPRESSION DEFINES A NOVEL RARE SUBSET OF

BURKITT LYMPHOMA

1) Characterize such BL MYC protein (Ve+/-Ve)

2) Evaluate whether there is cross talk with in the MYC gene family members in
BL.
3) Explore the genetic landscape of these rare BL cases (MYC-negative).
METHOD

University of Pathodiagnostike
Nairobi Lab, Berlino
KENYA GERMANY

University of
Moi University Siena
Eldoret ITALY
KENYA
University of
Firenze
ITALY
▪ IHC: rabbit monoclonal antibody Y69 (1:100). That produces a strict
nuclear staining with reproducible scores.
▪ FISH: Split-signal probes for IGH and Immunoglobulin light chain
gene - IGL loci (Vysis, Abbott Molecular IL, USA).
Fig. 1 A, B & C Frequency of MYC Translocation &
expressions in BL cases.
A. 81 (98%) cases were
positive by FISH & IHC.

B. 2 cases were negative by

FISH but express MYC
protein at variable level.

C. 7 cases were FISH

positive and IHC
negative.
Fig. 2 D

To investigate the effect of MYC absence by functional studies using MYC

RNA silencing in AKATA cell lines were performed

D) Silenced MYC gene results in higher expression of both MYCN mRNA and
protein.
 MYC RNA in the three groups
To correlate MYC protein expression with MYC gene presence at mRNA level using
qPCR

In the 7/90 (8%) cases lacking MYC protein expression, MYC mRNA levels
were significantly lower than those recorded in classic MYC-positive BL
(p=0.006), and lower than those observed in normal lymph-node in 4/7
(71%) cases.
MYC & MYCN is inversely correlated in BL

We then explored MYCN expression in the MYC-negative samples by

IHC. 6/7 MYC-negative cases showed clear MYCN expression at IHC
consistently.
 MYC & MYCN is inversely correlated in BL at mRNA
level

To investigate why MYC is not expressed in BL typical cases with t(8;14) we

looked at the genetic profile of the samples characterized by T+ and P-ve

✔ 6/7 MYC-negative cases showed significant levels of MYCN mRNA at qPCR

(p<0.001).
 Fig. 3 A, B, C & D The genetic profile of MYCN+ in BL is
different from MYC+/MYCN-Ve BL
Number of mutations/gene

We detected genes previously found to be

mutated in BL (MYC, EZH2, FGFR3,
RET, ARID1A, PARP1, PIK3R1,
NOTCH2, CDKN2A/B, and CDC73) in
addition to (ALK, EPHA3, EPHA7,
ETV1, IDH1, PBX1, PDGFRA/B, ROS1,
and TAL1).

✔ Ultra-deep targeted sequencing of 409 cancer associated genes, found 239

single nucleotide variants (SNVs), affecting 108 genes in 4 MYC-/MYCN+
cases.
 C) MYCN-ve cases presented more frequent SNVs in chromosomes
MYCN+ve presented an apparent opposite pattern (chi-square, p<0.0001)

✔ MYC and MYCN were affected by 97 and 37 SNVs, respectively; 33 and 13 were
predicted to be damaging for the protein structure in MYC and MYCN,
respectively.
✔ All cases presented with at least 9 SNVs in MYC and 3 in MYCN, including
at least 1 predicted as damaging.
✔ MYCN-Ve cases presented a remarkably lower number of SNVs distributed
across a small group of genes.
 Overall findings number 2

For the first time MYCN translocation and MYCN Protein

expression has been discovered in NHL.

❖ We therefore, describe a new entity of BL characterized by

the presence of MYCN translocation and MYCN protein
expressions that can replace the pathogenic role of MYC.

❖ The acquisition of somatic mutations can therefore, replace

EBV driven functions or contribution in tumor development.
Conclusions

EBV is probably involved NHL pathogenesis more often that currently

expected and known

Though MYC is the hallmark BL, a subtype exist that lacks MYC
expression but characterized MYCN expression

Therefore, EBV and MYC may cooperate in lymphogenesis of BL.

Whether confirmed on a larger cohort of cases and different tumor types,

the current study may support the rationale for strengthening the effort
toward EBV vaccination that could potentially prevent the development of
EBV‐associated neoplasms.
 R.Bob
M.R. Ambrosio H. Stain
S. Lazzi Pathodiagnostike lab, Berlino
G. Lo Bello GERMANY
L. Mundo
B.J. Rocca P. Oyiro
University of Siena J.Nyagol
ITALY N. Othieno-Abinya
University of Nairobi
KENYA
P.P. Piccaluga
University of Bologna
ITALY I.Ndede
K.Patel
M.R. Santi Moi University Eldoret
University of Firenze KENYA
ITALY
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