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Leukemia & Lymphoma
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ilal20
Fish evaluation of additional cytogenetic
aberrations and hyperdiploidy in childhood Burkitt
lymphoma
Georgia Avgerinou, Kalliopi Stefanaki, Konstantinos Liapis, Ioannis
V. Kostopoulos, Lydia Kossiva, Chryssa Tzoumaka-Bakoula, Dimitris
Pavlidis, Maria Filippidou, Katerina Katsibardi, Maria Ampatzidou, Antonis
Kattamis, Sophia Polychronopoulou, Marina Mantzourani & Stefanos I.
Papadhimitriou
To cite this article: Georgia Avgerinou, Kalliopi Stefanaki, Konstantinos Liapis, Ioannis V.
Kostopoulos, Lydia Kossiva, Chryssa Tzoumaka-Bakoula, Dimitris Pavlidis, Maria Filippidou,
Katerina Katsibardi, Maria Ampatzidou, Antonis Kattamis, Sophia Polychronopoulou, Marina
Mantzourani & Stefanos I. Papadhimitriou (2021): Fish evaluation of additional cytogenetic
aberrations and hyperdiploidy in childhood Burkitt lymphoma, Leukemia & Lymphoma, DOI:
10.1080/10428194.2021.1998480
To link to this article: https://doi.org/10.1080/10428194.2021.1998480
View supplementary material Published online: 02 Nov 2021.
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ORIGINAL ARTICLE
Fish evaluation of additional cytogenetic aberrations and hyperdiploidy in
childhood Burkitt lymphoma
Georgia Avgerinou
a
, Kalliopi Stefanaki
b
, Konstantinos Liapis
c,d
, Ioannis V. Kostopoulos
c,e
, Lydia Kossiva
f
,
Chryssa Tzoumaka-Bakoula
f
, Dimitris Pavlidis
c
, Maria Filippidou
a
, Katerina Katsibardi
a
, Maria Ampatzidou
g
,
Antonis Kattamis
a
, Sophia Polychronopoulou
g
, Marina Mantzourani
h
and Stefanos I. Papadhimitriou
c
a
Division of Pediatric Hematology-Oncology, First Department of Pediatrics, National and Kapodistrian University of Athens, “Aghia
Sophia”Children’s Hospital, Athens, Greece;
b
Department of Pathology, “Aghia Sophia”Children’s Hospital, Athens, Greece;
c
Department of Laboratory Hematology, “G.Gennimatas”Athens General Hospital, Athens, Greece;
d
Department of Hematology,
Alexandroupolis University Hospital, Democritus University of Thrace, Alexandroupolis, Greece;
e
Department of Biology, School of
Science, National & Kapodistrian University of Athens, Athens, Greece;
f
Second Department of Paediatrics, School of Medicine, “P. &
A. Kyriakou”Children’s Hospital, National and Kapodistrian University of Athens (NKUA);
g
Department Of Paediatric Haematology-
Oncology, “Aghia Sophia”Children’s Hospital Athens, Greece;
h
Department of Internal Medicine, School of Medicine, National and
Kapodistrian University of Athens, Laikon General Hospital, Athens, Greece
ABSTRACT
Beyond MYC rearrangement, Burkitt lymphoma (BL) often presents with additional aberrations.
Biopsy touch imprints from 72 children with BL were tested with interphase fluorescence in-situ
hybridization (i-FISH) for MYC,BCL2,BCL6, IGH, IGK and IGL rearrangements and copy-number
aberrations involving 1q21/1p32, 7cen/7q31, 9cen/9p21, 13q14/13q34 and 17cen/17p13. Diploid
status deviations were investigated with chromosome enumeration probes. MYC rearrangement
was demonstrated in all cases. Additional aberrations included þ1q (21/72:29.2%), þ7q (14/
72:19.4%), 13q- (14/72:19.4%), 9p-(6/72:8.3%) and hyperdiploidy (6/72:8.3%). Advanced clinical
stage IV, þ7q and 9p- were associated with shorter overall survival, with stage IV and þ7q
retaining prognostic significance on multivariate analysis. No relapse or death was reported
among the hyperdiploid cases. This i-FISH investigation provides information on the genetic pro-
file of BL and may prove valuable for patients with no karyotype analysis. Demonstration of
hyperdiploidy could evolve research on clonal evolution pathways and probably identify a sub-
group of children with favorable prognosis.
ARTICLE HISTORY
Received 8 April 2021
Revised 2 October 2021
Accepted 11 October 2021
KEYWORDS
FISH; hyperdiploidy;
children; Burkitt lymphoma;
cytogenetics
Introduction
Burkitt lymphoma (BL) is an aggressive mature B-cell
neoplasia affecting patients of all ages. It is the most
common lymphoma in childhood accounting for
40–50% of pediatric cases. While the disease is now
curable in most children, it is still associated with a
poorer prognosis in adult patients [1].
Nearly all BL cases harbor rearrangement of MYC
oncogene on 8q24, in the context of a reciprocal
translocation involving the immunoglobulin heavy-
chain locus, on 14q32, or, less often, the light-chain
loci. This results in the upregulation of MYC expres-
sion, disrupting the cell cycle and proliferation control.
The translocation t(8;14)(q24;q32) is found in 70–80%,
with its variants, t(2;8)(p12;q24) and t(8;22)(q24;q11),
observed in 10–15% of patients [2]. Though BL usually
presents with simple karyotype [1], it was long ago
recognized that 8q24 translocation may be accompa-
nied by nonrandom additional chromosome aberra-
tions leading to a higher degree of genetic complexity
[3–8], a finding confirmed by recent genome-wide
investigations which revealed multiple molecular
lesions [9–13]. While some authors have suggested
that these genetic lesions are essential in lymphoma-
genesis, others believe that they derive from clonal
evolution which parallels the disease progression,
since they are often seen in sequential karyotyping of
relapsed cases [14] or are detected in cultures of
tumor cells in vitro [15]. Several groups have investi-
gated the clinical significance of additional aberrations
in childhood BL, though not with consistent conclu-
sions [16–22].
CONTACT Georgia Avgerinou g.avgerinou@yahoo.gr Division of Pediatric Hematology-Oncology, First Department of Pediatrics, National and
Kapodistrian University of Athens, ‘Aghia Sophia’Children’s Hospital Thivon and Papadiamantopoulou, Athens 11527, Greece
Supplemental data for this article can be accessed here.
ß2021 Informa UK Limited, trading as Taylor & Francis Group
LEUKEMIA & LYMPHOMA
https://doi.org/10.1080/10428194.2021.1998480
In some cases, cytogenetic investigation has shown
hyperdiploidy, often of a very high (‘near-tetraploid’)
level [23]. However, to the best of our knowledge, the
potential biological and clinical implications of hyper-
diploidy in pediatric BL have not been investigated so
far. We have investigated the significance of additional
chromosome aberrations in a large group of children
with BL, including hyperdiploid cases revealed by the
application of interphase fluorescence in-situ hybrid-
ization (i-FISH), in the context of routine diagnostic
work-up.
Patients, materials and methods
Patients
The study included 72 children (55 boys), aged
2–16 years (median 9), diagnosed, and treated in
‘Aghia Sofia’Children’s Hospital over a ten-year period
(January 2010–June 2019). All children were previously
immunocompetent and had a definite diagnosis of BL,
based on both histology and demonstration of MYC
rearrangement, shown by either conventional cytogen-
etics or i-FISH. Patients underwent complete staging,
including examination of bone marrow and cerebro-
spinal fluid and were treated according to NHL-BFM or
FAB-LMB regimen (44 and 28 patients, respectively).
The main demographic and clinical data are shown on
Table 1. Ethical approval was obtained from the insti-
tutional review board and written informed consent
was provided by parents or guardians according to
the Declaration of Helsinki.
Tissue samples
In all cases, touch imprints were prepared from fresh
biopsy samples of involved sites (mainly abdominal
masses or lymph nodes). Application of May-
Gr€
unwald-Giemsa stain confirmed infiltration of all
imprints with cells showing L3-morphology.
Immediately upon preparation, imprints were fixed in
methanol: acetic acid 3:1 and subsequently stored at
20C until utilized for FISH study.
Cytogenetic investigation
An extensive i-FISH study was performed on the touch
imprints, employing commercially available probes
(Abbott Molecular Inc, Des Plaines, Ill, USA; Cytocell
Ltd, Cambridge, UK). The investigation was completed
in three steps (Supplementary Table 1). Initially, all
patients suspected of BL were tested for the
rearrangement of MYC,BCL2,BCL6, IGH, IGK and IGL
genes and the MYC/IGH fusion, with a probe set
including a centromeric probe (cen) for chromosome
8. Secondarily, in all cases with MYC rearrangement,
application of probes targeting 1q21/1p32, 7cen/7q31,
9cen/9p21 (INK4 locus), 13q14 (D13S319)/13q34 and
17cen/17p13 (TP53) was performed. Finally, cases with
any signal pattern indicating deviation from the dip-
loid status were further investigated for a numerical
aberration of the respective chromosome, with add-
itional use of a centromeric (chromosomes 1, 2, 3 and
18) or another chromosome enumeration probe.
Particularly, chromosome 14 was investigated with
TCRAD break-apart probe, since the gene is located
on 14q11, close to the centromere, and is normally
expected to be unrearranged in B-cell malignancy. For
chromosome 22, the number of signals for the IGL
gene was considered as a chromosome enumerator
(taking into account only the proximal probe in cases
with IGL rearrangement). No additional enumerator
was used for chromosome 13. Thus, this line of testing
allowed for definitive enumeration of chromosomes 1,
2, 3, 7, 8, 9, 17 and 18 and provided indirect clues for
the status of chromosomes 13, 14 and 22. For the pur-
poses of the study, hyperdiploidy was assumed based
on at least three chromosomes over-represented, as
shown with the use of centromeric probes, without
evidence of any monosomy.
All probes were applied following manufacturer’s
instructions for cytological preparations and results
were based on the observation of at least 200 clearly
defined, non-overlapping nuclei. Cutoff values for
false-positive results had already been established in
our laboratory by applying the respective probe on
lymph node or other solid tissue imprints from
patients with non-neoplastic disorders (Supplementary
Table 1).
In patients with bone marrow infiltration, conven-
tional cytogenetic study was performed on anticoagu-
lated marrow samples after unstimulated culture of
24, 48 and 72 h.
Statistics
Categorical variables were summarized by counts and
percentages and continuous variables by medians and
ranges were compared between groups with the use
of Fisher’s exact test and Mann-Whitney-U test,
respectively. The primary endpoint was overall survival
(OS), i.e. the time from diagnosis to death due to any
cause. Impact on outcome was assessed with Kaplan-
Meier curves and log-rank test. Multivariate analysis
was performed with the Cox proportional hazard
2 G. AVGERINOU ET AL.
Table 1. Main demographic, clinical and cytogenetic data for the children enrolled.
Patient id Sex
Age
(yrs) Stage Outcome
OS
(mos)
MYC
Rearrangement 1q21/1p32 7cen/7q31 9cen/9p21 13q14/13q34 17cen/17p13 HRD
#1 F 11 III A 117 MYC/IGH 3 1q21, 2 1p32 Normal Normal Normal Normal No
#2 M 13 III A 114 MYC/IGH Normal Normal Normal Normal Normal No
#3 M 11 IV A 112 MYC/IGH Normal Normal Normal Normal Normal No
#4 M 9 III A 112 MYC/IGH Normal Normal 2 9cen, 1 9p21 Normal Normal No
#5 M 14 III A 109 MYC/IGH 3 1q21, 2 1p32 Normal Normal Normal Normal No
#6 M 4 III A 106 MYC/IGH Normal Normal Normal Normal Normal No
#7 M 7 II A 105 MYC/IGH Normal Normal Normal 2 13q14, 1 13q34 Normal No
#8 F 9 III A 103 MYC Normal Normal Normal Normal Normal No
#9 M 4 III A 100 MYC/IGH 3 1q21, 2 1p32 Normal Normal Normal Normal No
#10 M 14 II A 97 MYC/IGH 3 1q21, 2 1p32 Normal Normal Normal Normal No
#11 F 4 III A 95 MYC/IGH Normal 3 7cen, 3 7q31 Normal Normal 3 17cen, 3 17p13 Yes
#12 M 14 III A 92 MYC þIGKþNormal Normal Normal Normal Normal No
#13 M 14 III A 89 MYC/IGH Normal Normal Normal 4 13q14, 4 13q34 Normal No
#14 M 6 III R/D 7 MYC/IGH 3 1q21, 2 1p32 2 7cen, 3 7q31 1 9cen, 1 9p21 3 13q14, 3 13q34 Normal No
#15 M 8 II A 87 MYC/IGH Normal Normal Normal 3 13q14, 3 13q34 Normal No
#16 M 4 III A 87 MYC/IGH 3 1q21, 2 1p32 Normal Normal Normal Normal No
#17 M 16 IV A 85 MYC/IGH 3 1q21, 2 1p32 Normal Normal Normal Normal No
#18 M 15 IV A 82 MYC/IGH Normal Normal Normal Normal Normal No
#19 M 9 III A 79 MYC/IGH Normal Normal Normal Normal 3 17cen, 3 17p13 Yes
#20 M 5 IV R/D 10 MYC/IGH Normal Normal 2 9cen, 1 9p21 Normal Normal No
#21 M 3 III A 78 MYC/IGH Normal Normal 2 9cen, 1 9p21 Normal Normal No
#22 F 15 IV A 76 MYC/IGL Normal Normal Normal Normal Normal No
#23 F 14 II A 73 MYC/IGH 3 1q21, 2 1p32 Normal Normal Normal Normal No
#24 F 11 II A 72 MYC Normal Normal Normal Normal Normal No
#25 F 14 II A 70 MYC/IGH Normal Normal Normal 2 13q14, 1 13q34 Normal No
#26 M 15 III A 70 MYC/IGH Normal Normal Normal 2 13q14, 1 13q34 Normal No
#27 M 4 III A 68 MYC/IGH Normal 3 7cen, 3 7q31 Normal 2 13q14, 1 13q34 Normal No
#28 M 12 III A 67 MYC/IGH Normal Normal Normal Normal Normal No
#29 M 9 III A 63 MYC/IGH 3 1q21, 2 1p32 Normal Normal Normal Normal No
#30 M 7 III A 60 MYC/IGH Normal Normal Normal Normal Normal No
#31 M 3 IV Rf/D 18 MYC/IGH Normal 2 7cen, 3 7q31 Normal Normal 2 17cen, 1 17p13 No
#32 F 11 III A 60 MYC/IGH Normal 3 7cen, 3 7q31 Normal 4 13q14, 4 13q34 3–417cen, 3–417p13 Yes
#33 M 7 III A 59 MYC/IGH Normal Normal Normal Normal Normal No
#34 M 3 III A 56 MYC/IGH Normal Normal Normal Normal Normal No
#35 M 4 III A 56 MYC þIGLþ31q21, 2 1p32 2 7cen, 3 7q31 Normal Normal Normal No
#36 M 11 III A 54 MYC/IGH Normal Normal Normal Normal Normal No
#37 M 2 III A 53 MYC/IGH Normal Normal Normal Normal Normal No
#38 F 12 III A 53 MYC/IGH Normal Normal Normal 2 13q14, 1 13q34 Normal No
#39 M 4 IV A 51 MYC/IGH 4 1q21, 2 1p32 Normal Normal Normal Normal No
#40 M 4 III A 50 MYC þIGKþ31q21, 2 1p32 Normal Normal Normal Normal No
#41 M 4 III A 48 MYC/IGH Normal Normal Normal Normal Normal No
#42 F 11 III A 45 MYC/IGH 3 1q21, 2 1p32 Normal Normal Normal Normal No
#43 M 9 III A 43 MYC/IGH Normal Normal Normal Normal Normal No
#44 M 15 III A 41 MYC/IGH Normal Normal Normal 2 13q14, 1 13q34 Normal No
#45 M 11 III Rf/D 15 MYC þIGLþNormal 2 7cen, 3 7q31 Normal Normal Normal No
#46 M 4 III A 39 MYC/IGH 3 1q21, 2 1p32 Normal Normal Normal Normal No
#47 M 5 III A 37 MYC/IGH 3 1q21, 2 1p32 3 7cen, 3 7q31 3 9cen, 3 9p21 3 13q14, 3 13q34 3 17cen, 3 17p13 Y
#48 M 4 II A 36 MYC/IGH Normal Normal Normal Normal Normal No
#49 M 9 III A 34 MYC/IGH Normal Normal 2 9cen, 1 9p21 Normal Normal No
(continued)
FISH IN CHILDHOOD BURKITT LYMPHOMA 3
Table 1. Continued.
Patient id Sex
Age
(yrs) Stage Outcome
OS
(mos)
MYC
Rearrangement 1q21/1p32 7cen/7q31 9cen/9p21 13q14/13q34 17cen/17p13 HRD
#50 F 12 III A 34 MYC/IGH 3 1q21, 2 1p32 Normal Normal Normal Normal No
#51 M 12 III A 33 MYC/IGH Normal 3 7cen, 3 7q31 Normal 3 13q14, 3 13q34 3 17cen, 3 17p13 Y
#52 M 12 II A 33 MYC Normal Normal Normal 2 13q14, 1 13q34 Normal No
#53 M 8 III A 32 MYC/IGH Normal Normal Normal 2 13q14, 1 13q34 Normal No
#54 M 5 II A 30 MYC/IGH Normal Normal Normal Normal Normal No
#55 F 4 II A 30 MYC þIGLþNormal Normal Normal 1 13q14, 2 13q34 Normal No
#56 M 9 II A 29 MYC þIGKþNormal Normal Normal Normal Normal No
#57 M 10 III A 27 MYC/IGH 3 1q21, 2 1p32 Normal Normal Normal Normal No
#58 F 9 IV Rf/D 10 MYC/IGH Normal Normal Normal Normal Normal No
#59 M 9 III A 26 MYC/IGH Normal Normal Normal Normal Normal No
#60 M 3 II A 24 MYC þIGLþNormal 2 7cen, 3 7q31 Normal Normal Normal No
#61 M 9 III A 20 MYC/IGH Normal Normal Normal 1 13q14, 1 13q34 Normal No
#62 M 6 II A 19 MYC/IGH Normal Normal Normal Normal Normal No
#63 F 16 IV R/D 18 MYC/IGK 3 1q21, 1 1p32 2 7cen, 3 7q31 Normal Normal Normal No
#64 M 13 III R/D 14 MYC/IGH 4 1q21, 2 1p32 2 7cen, 3 7q31 Normal 2 13q14, 1 13q34 Normal No
#65 F 14 IV A 17 MYC/IGH Normal Normal Normal Normal 4 17cen, 4 17p13 Y
#66 M 8 II A 14 MYC/IGH Normal Normal Normal Normal Normal No
#67 M 16 IV R/D 6 MYC/IGH Normal 2 7cen, 3 7q31 Normal 2 13q14, 1 13q34 Normal No
#68 M 14 III A 13 MYC/IGH Normal Normal Normal 2 13q14, 1 13q34 Normal No
#69 F 13 II A 11 MYC/IGH 3 1q21, 2 1p32 2, 7cen, 3 7q31 Normal Normal Normal No
#70 F 7 III A 9 MYC/IGH Normal Normal Normal Normal Normal No
#71 M 13 IV A 8 MYC/IGH 3 1q21, 2 1p32 Normal 2 9cen, 1 9p21 Normal Normal No
#72 M 15 III A 7 MYC/IGH Normal Normal Normal Normal Normal No
M: male; F: female; yrs: years; A: alive; R: relapse; Rf: refractory disease; D: death; OS: overall survival; mos: months; HRD: hyperdiploidy. Details on the cytogenetic features of hyperdiploid cases are shown on
Table 3 and Supplementary Table 2.
4 G. AVGERINOU ET AL.
model. Significance for all tests was assumed at
p<.05. All analyses were done on the IBM SPSS statis-
tical software (version 26.0) for Windows.
Results
MYC rearrangement
In all BL cases, rearrangement of MYC gene was dem-
onstrated by i-FISH with application of the break-apart
probe used. The MYC/IGH fusion was found in 60
patients (83.3%). In 4 and 5 cases (5.6% and 6.9%), the
test for MYC/IGH was negative, but the IGK or IGL was
found rearranged. In 2 of them (patients #63 and #22),
the marrow karyotype showed the presence of t(2;8)
and t(8;22) (Table 2). The remaining 7 cases, for which
a karyotype was not available, were assumed to har-
bor MYC/IGK or MYC/IGL fusion, but since definite
proof was lacking, they were reported as
MYCþIGK þor MYCþIGLþ, respectively (Table 1).
Finally,in3cases(#8,#24and#52;Table 1), a partner
gene for the rearranged MYC could not be identified,
since karyotype was not available and i-FISH tests for
MYC/IGH and IGK or IGL rearrangement were all negative.
Additional chromosomal aberrations
In 47 cases (65.3%), at least one additional aberration
was revealed by either conventional cytogenetics or
i-FISH (Table 1;Supplementary Table 2). The most
common one was 1q gain (þ1q21), found in 21
patients (29.2%), without evidence of total overrepre-
sentation of chromosome 1. A 7q gain (þ7q31) was
detected in 14 cases (19.4%), 5 of whom showed total
trisomy 7. A 13q deletion was shown in 14 cases
(19.4%), involving 13q14, 13q34 or both regions in 2,
11 and 1, respectively. A heterozygous 9p loss was
found in 6 patients (8.3%), one of whom with evi-
dence of monosomy 9. Finally, 17p deletion was found
in one (1.4%) patient only.
No case was found with either BCL6 or BCL2
rearrangement. However, in one patient only one
intact BCL6 gene was demonstrated (without mono-
somy 3), suggesting a 3q27 deletion. Similarly, two
patients with three intact BCL2 genes were found with
trisomy 18 and in two cases trisomy 8 was demon-
strated (Supplementary Table 2). The latter 4 cases did
not fulfill the hyperdiploidy criteria set in the study.
Concordance between karyotypic and
i-FISH findings
Conventional cytogenetic investigation was successful
in all 12 children with bone marrow involvement,
showing 8q24 translocation in all cases and hyperdi-
ploidy in only one (patient #65) (Table 2). There were
no major discrepancies between karyotypes and
Table 2. G-banded karyotypes for patients with bone marrow infiltration.
Patient ID Marrow karyotype
#3 45,X,-Y,t(8;14)(q24;q32)[14]/46,XY[11]
#17 46,XY,dup(1)(q21q32),t(8;14)(q24;q32)[18]/46,XY[2]
#18 46,XY,t(8;14)(q24;q32)[10]/46,XY[10]
#20 46,XY,t(8;14)(q24;q32),þ12, –20[13]/46,XY[7]
#22 46,XX,t(8;22)(q24;q11)[20]
#31 46,XY,t(8;14)(q24;q32),der(17)t(?;17)(?;p11)[22]/46,XY[3]
#39 46,XY,dup(1)(q21q31),t(8;14)(q24;q32)[7]/46,XY,idem,der(18)t(1;18)(q11;p11)[10]/46,XY[8]
#58 46,XX,t(8;14)(q24;q32)[25]/46,XX[4]
#63 44,XX,i(1)(q10),t(2;8)(p12;q24), –4,der(6)t(6;7)(q23;q21), –10,del(10)(q23)[20]
#65 56,XXX,þ4,þ6,þ8,t(8;14)(q24;q32),þ10,þder(14)t(8;14),þ17,þ17,þ18,þ21[20]/46,XX[5]
#67 46,XY,t(8;14)(q24;q32)[3]/46,XY,idem,der(13)t(7;13)(q11;q34)[11]/46,XY[8]
#71 46,X,-Y,del(6)(q21q26),t(8;14)(q24;q32),þder(?)t(1;?)(q21;?)[12]/46,X,-Y,idem,t(17;18)(q24;q21)[13]
The number of aberrant and normal mitoses is indicated in brackets.
Table 3. The degree of involvement of the chromosomes tested in hyperdiploid cases. Blank cells represent normal findings.
Patient
ID
Chromosomes
Number of
chromosomes
involved
Tested with centromeric probes Tested with other enumerators
1 2 3 7 8 9 17 18 13q14/13q34
14q11
(TCRAD)
22q11
(IGL)
#11 x3 x3 x3 x3 x3 5
#19 x3 x3 x3 x3 x3 3
#32 x4 x3 x4 x3,x4 x4,x5 4 13q14, 4 13q34 x3 x3 5
#47 x3,x4 x3 x3,x4 x3 x3 x4 3 13q14, 3 13q34 x3 6
#51 x3 x3 x3 x3 x3 x4 313q14, 3 13q34 6
#65 x3 x4 x3 x3 3
The estimation of the number of chromosomes involved is based on the over-representation of chromosomes tested with centromeric probes only.
FISH IN CHILDHOOD BURKITT LYMPHOMA 5
respective i-FISH findings. A single exception was
patient #20, in whom i-FISH showed 9p21 deletion,
not demonstrated or suggested in the karyotype.
Hyperdiploid cases
In 6 cases (8.3%), i-FISH findings fulfilled hyperdiploidy
criteria (Table 3). According to i-FISH, the number of
chromosomes definitely involved ranged from 3 to 6.
Yet, in patient #65 the karyotype revealed 9 chromo-
somes overrepresented, against 3 shown by i-FISH.
The commonest pattern was trisomy, but tetrasomies
and pentasomies were also noted. In two patients
(#32 and #47; Table 3), some of the excessive chromo-
somes were represented at a variable level. Among
tested chromosomes, those involved in all cases were
chromosomes 17 and 18, while chromosomes 3, 8 and
7 were found supernumerary in 5, 5 and 4 cases
respectively. In contrast, there was no evidence for any
numerical aberration of chromosome 1, either among
the hyperdiploid or the non-hyperdiploid cases.
Except for patient #47, in whom 1q gain was docu-
mented, there was no evidence for any structural
aberration among the hyperdiploid cases. All 6 of
these patients were found with MYC/IGH fusion. A
careful evaluation of the signal pattern in the tests for
MYC/IGH and MYC and IGH rearrangement ruled out
the possibility for coexistence of hyperdiploid and
non-hyperdiploid subclones, showing that the neo-
plastic population consisted of hyperdiploid cells only.
Correlation with outcome
During the follow-up period (median 50.5 months/
range 7–117), 8 children succumbed to the lymphoma,
3 of them with refractory disease and 5 after relapse.
The median survival of these 8 children was
14.5 months (6–18). As of January 2020, the remaining
64 children were alive and in complete remission.
Among age, clinical stage, type of MYC rearrange-
ment and the main additional aberrations, only stage
IV, 7q gain (but not trisomy 7) and 9p loss were sig-
nificantly associated with outcome. Particularly, OS
was significantly different for stage IV patients (median
not reached/range 6–112 months), compared to both
stage II (not reached/11–105; Hazard Ratio: 14.66, 95%
Confidence Intervals: 2.30–92.01; p¼.0042) and stage
III (not reached/7–117; HR: 7.6, 95% CI: 1.25–46.53;
p¼.009) or stages II and III combined (HR: 10, 24, 95%
CI: 1.39–75.42; p<.0001) (Figure 1(A,B)). The effect of
additional chromosome abnormalities on OS is graph-
ically depicted on Figure 1(C). Compared with the
group with an 8q24 translocation as the sole finding
(median not reached/range 7–114 months), children
with a 7q gain (but not trisomy 7) experienced an
inferior outcome (18/6–56; HR: 23.3 95% CI:
3.72–146.2; p<.0001) and the same was observed for
children with tumors bearing –9/9p- (not reached/
7–112; HR: 10.5, 95% CI: 0.48–230.9; p¼.017). In the
multivariate analysis, 7q gain (versus sole MYC
rearrangement, HR: 16.67, 95% CI: 3.08–90.9; p<.001)
and stage IV (versus II and III combined, HR: 2.13, 95%
CI: 1.01–4.54; p¼.049) retained significance as inde-
pendent prognostic factors. There was no relapse or
death observed among the hyperdiploid cases and
overall survival was comparable to children with
t(8q24) only.
Discussion
In this study we have applied i-FISH for the initial
cytogenetic investigation of childhood BL, with the
use of a broad panel of probes. Although this
approach does not provide a general overview of the
whole chromosome set, it can detect essential abnor-
malities, like gene rearrangements, copy-number aber-
rations of specific regions and –if supplemented with
the use of the appropriate chromosome enumeration
probes –whole chromosome aneusomies. Obviously,
the technique is particularly useful for the diagnostic
approach of the majority of children without bone
marrow infiltration, in which a karyotype is not easy to
obtain. To our knowledge, this is the largest extensive
i-FISH study of childhood BL so far.
Though BL is generally considered to initially pre-
sent with simple karyotype, our results revealed add-
itional chromosome aberrations in nearly two thirds
(65.3%) of our patients at diagnosis. This finding sup-
ports the results of earlier studies on previously
untreated pediatric patients, in which the rate ranged
between 64% and 89% [12,17–19], reaching almost
100% when whole-genome molecular techniques were
applied to detect small scale imbalances [13].
Although the reasons and pathogenic significance of
such chromosomal diversity at diagnosis remain
obscure, it cannot be interpreted solely based on the
genomic instability associated with the neoplastic pro-
cess. From a practical point of view, it indicates that
–except for BCL2 and/or BCL6 rearrangements–the
presence of additional aberrations should not rule out
the diagnosis of BL.
Several studies have demonstrated that certain
chromosome lesions represent recurrent secondary
aberrations in BL [3–6,8–13,16–20]. In our targeted
6 G. AVGERINOU ET AL.
investigation, þ1q, þ7q and 13q- were detected with
frequencies largely comparable to those previously
reported. It is noteworthy that the probes we used for
i-FISH assays were not designed specifically for BL. For
example, 1q21/1p32 set is primarily intended for the
investigation of multiple myeloma [24]. However, it is
appropriate in detecting 1q gains in BL too, since in
pediatric cases the chromosomal region involved in
þ1q appears to be rather large [12] and almost always
includes 1q21 [9,10,12]. The same holds true for 7cen/
7q31 probe, widely employed for the detection of –7/
7q- in myeloid malignancies. While some authors have
suggested that the minimally involved region is
7q32–q36 [9,16], it seems that þ7q in BL is a large
scale aberration [19], steadily involving 7q31 [10,13].
Similarly, the 13q14/13q34, routinely used for the
study of chronic lymphocytic leukemia and similar
lymphoproliferations [25], is equally effective in detect-
ing 13q losses in BL, even when the aberration is
missed in the karyotype [20]. It may not, however,
detect 13q gains centered on the 13q32 region
[12,17]. It is interesting that, while previous studies in
smaller pediatric cohorts have found 17p- in as many
as 15% of the patients [12], we have detected it in
only one of our 72 children, thus supporting the view
that 17p- is a recurrent additional aberration in adult
rather than childhood BL [9]. Finally, 9p21 loss, found
in 8.3% of our patients, is not frequently mentioned as
a secondary aberration in BL. This is perhaps related
to the method applied for its detection. It has been
reported that in acute lymphoblastic leukemia (ALL)
the incidence of 9p- investigated by FISH is up to four
times higher than that obtained with conventional
cytogenetics [26]. The situation may be similar in BL:
among 182 karyotypes of affected children reported
by Poirel et al., the rate of those with any aberration
resulting in 9p- was 3.8% [19], whereas in smaller
groups studied with application of high resolution
molecular techniques the respective rate was 10%
(among patients of all ages) to 15.4% (among pediat-
ric patients only). In our series, only two children with
9p21 deletion detected by i-FISH had a karyotype
available. In one of them (patient #20), the aberration
was apparently missed in the banding analysis. In the
other one (patient #71), the karyotype showed an
unidentified derivative chromosome which could rep-
resent a chromosome 9 with part of the short
arm deleted.
Figure 1. Impact of clinical stage (A,B) and additional chromosome aberrations (C) on overall survival (OS).
FISH IN CHILDHOOD BURKITT LYMPHOMA 7
The most common secondary aberrations have
been evaluated as potential prognostic markers
[16–20], but the results were contradictory. The con-
troversies may be due to several factors, including lim-
ited number of children, preferential enrollment of
advanced stage disease and mixed cohorts of children
and adults. Moreover, some investigators relied exclu-
sively on conventional cytogenetic analysis while others
supplemented their investigation with FISH techniques.
The heterogeneity of the aberration evaluated may
have also led to conflicting results. One study con-
cluded that all 13q abnormalities confer adverse prog-
nosis [18], but another limited the range of clinically
relevant lesions to deletions only [19]. Similarly, Lones
et al. suggested that the critical region is 13q32 [17],
while Nelson et al. favored 13q14 deletions as the
prognostically crucial event [20]. Finally, since childhood
BL is now curable in most cases, a common problem in
evaluating prognostication is the limited number of
children experiencing an adverse outcome.
In our cohort, the overall fatality rate was 11.1%,
with 8 of the 72 children succumbing within
18 months from diagnosis. Apart from the clinical
stage, significant association with OS was found only
for þ7q31 and –9/del(9p21). Of note, in nearly one
third (5 of 14) of our cases with þ7q the aberration
appeared as the result of total trisomy 7, but the
prognostic significance referred only to patients with
an unbalanced 7q gain and was not retained when
cases with þ7 were included in the analysis.
Regarding this distinction, previous studies have pro-
vided diverging results. Although Poirel et al. observed
that half of the children with BL and a 7q gain had an
underlying trisomy 7, they concluded that the whole
subgroup with þ7/þ7q was at a higher risk for dismal
outcome [19]. On the contrary, Nelson et al. did not
find any prognostic value for þ7, investigated with
both G-banding and FISH [20]. It should be remarked
that 4 of our 5 patients with þ7 presented also with
hyperdiploidy, which may modify the clinical signifi-
cance of individual aberrations, as discussed below.
9p21 deletions have been reported in all types of
aggressive and chronic lymphoid malignancies. Yet, to
our knowledge, they have not been evaluated as a
prognostic factor in BL, perhaps due to the difficulty
in detecting the aberration by conventional cytogenet-
ics, as explained above. The deletion results in the loss
of the tumor suppressors CDKN2A and CDKN2B.
CDKN2A is transcribed into either p16
INK4A
or p14
ARF
protein, under the control of different promoters [27].
Of these, p16
INK4A
is a cyclin-dependent kinase inhibi-
tor that blocks the action of cyclin D-dependent
kinase, inducing cell cycle arrest. The loss or deficiency
of the protein function has also been implicated in
the enhancement of various oncogenes [28], inhibition
of apoptosis [29], and promotion of chemoresistance
[30]. Though 9p21 deletion is not unanimously
accepted as an independent risk factor in childhood
ALL, it has been found to further deteriorate prognosis
in already highly aggressive disease, as in Ph þcases
[31], even in the era of tyrosine kinase inhibitors [32].
Moreover, it may modify the effect of favorable gen-
etic features, such as the ETV6/RUNX1 fusion [33,34].
Finally, in BL CDKN2A promoter is frequently found
hypermethylated which, in combination with an het-
erozygous deletion, results in biallelic inactivation of
the gene [35]. Although it did not emerge as an inde-
pendent prognostic factor in our study, we believe
that –9/del(9p21) deserves to be further investigated
in BL, both in terms of incidence and clinical
significance.
The application of enumeration probes for certain
chromosomes enabled us to identify 6 hyperdiploid
cases among the 72 children enrolled in our study
(8.3%), with at least 3 chromosomes definitely
involved in each case. Thus, in the absence of indica-
tions for any monosomy, it is reasonable to assume
that these cases displayed hyperdiploidy at about the
level of 50 chromosomes. Obviously, since our criter-
ion for hyperdiploidy was the over-representation of
at least three chromosomes, cases with hyperdiploidy
of a lower level or with involvement of chromosomes
other than the ones targeted were not recognized. We
are not aware of any previous attempt to assess the
frequency of hyperdiploidy in BL. Among 182 karyo-
types listed by Poirel et al., the number of those with
any modal number above 46 was 44 (24.2%), while
there were only 4 cases (2.2%) with at least 50 chro-
mosomes [19]. In the Mitelman database, among
patients up to 16-year-old the respective rates are
24.4% and 2.6% [23], thus indicating that hyperdi-
ploidy in BL is less frequent than in childhood ALL.
Since we have not tested chromosome 21 or the sex
chromosomes, we cannot draw any conclusion regard-
ing the chromosomes commonly involved in compari-
son to the situation in ALL. However, chromosomes 3
and 7 found trisomic or tetrasomic in 5 and 4 of our
six hypediploid cases respectively are not often
involved in the hyperdiploidy of ALL. Although there
were limited variations regarding the level of over-rep-
resentation of certain chromosomes in two of the six
patients, there was no evidence for coexistence of
non-hyperdiploid and hyperdiploid subclones within
the cellular neoplastic population, suggesting that in
8 G. AVGERINOU ET AL.
BL hyperdiploidy usually results from a fast clonal evo-
lution process which is completed by the time
of diagnosis.
Interestingly, there were no cases of refractory dis-
ease, relapse or death among our patients with hyper-
diploid tumors. Their OS was comparable to that of
the subgroup with rearranged MYC only. Given the
small number of hyperdiploid cases in our cohort, this
may well be a matter of chance. Alternatively, it may
signify that hyperdiploidy is indeed associated with
favorable prognosis in BL. In ALL the beneficial effect
of hyperdiploidy has been attributed to various mech-
anisms, i.e. a tendency of the malignant cells toward
apoptosis [36,37] or increased intracellular accumula-
tion of methotrexate metabolites [38]. Moreover, it has
been suggested that hyperdiploidy may counterbal-
ance the deregulated expression of oncogenes and
tumor suppressors associated with disease progression
by providing new copies of unaffected alleles. In sup-
port of this assumption is the observation that hyper-
diploidy ameliorates the dismal outcome associated
with high-risk karyotypes in other diseases, like ALL
[30] and multiple myeloma [39]. Clearly, additional
investigation is warranted to demonstrate whether in
childhood BL hyperdiploidy merely reflects genetic
instability or truly confers a favorable prognosis.
In conclusion, we have applied a wide panel of
probes for cytogenetic study of children with BL and
showed that this approach may provide clinically
meaningful information for patients without a karyo-
type available, by revealing MYC rearrangement and
additional prognostically important aberrations. The
demonstration of hyperdiploidy using chromosome
enumeration probes may offer the means of better
understanding the pathways of clonal evolution and,
perhaps, help toward the recognition of a low-risk
subgroup of patients who could benefit from low-
intensity chemotherapy regimens, reducing the risk of
late adverse effects and improving the prospects of
long-term survivors from this curable disease.
Originality statement
This manuscript contains original material that has not
been published or submitted previously to another
journal. All authors agree to the submission of this
manuscript to Leukemia & Lymphoma.
Patient consent
The patients’parents or guardians have provided writ-
ten informed consent for this study.
Ethical approval
All clinical samples and data were collected for routine
patient care. This retrospective study was done in
accordance with the ethical standards of the
Institutional Research Committees of ‘Georgios
Gennimatas’Hospital and ‘Aghia Sofia’Children’s
Hospital, and in compliance with the ethical principles
of the Declaration of Helsinki.
Disclosure statement
No potential conflict of interest was reported by
the author(s).
Funding
The author(s) reported there is no funding associated with
the work featured in this article.
ORCID
Antonis Kattamis http://orcid.org/0000-0002-5178-0655
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FISH IN CHILDHOOD BURKITT LYMPHOMA 11
