CREBBP IS A MAJOR PROGNOSTIC BIOMARKER FOR RELAPSE IN CHILDHOOD B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA: A NATIONAL STUDY OF UNSELECTED COHORT
Krstevska Bozhinovikj E1, Matevska-Geshkovska N1, Staninova Stojovska M1,
Gjorgievska E1, Jovanovska A2, Kocheva S2*, Dimovski A1,3*
*Corresponding Author: *Corresponding Authors: Prof. Aleksandar Dimovski MD PhD. Center for Biomolecular Pharmaceutical
Analyses, Faculty of Pharmacy, University Ss. Cyril and Methodius in Skopje, Mother Theresa 47, 1000
Skopje, N. Macedonia adimovski@ff.ukim.edu.mk; phone number: +38923119694 ext109;
Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Macedonian Academy
of Sciences and Arts, Bul. Krste Misirkov 2, 1000, Skopje, N. Macedonia, a.dimovski@manu.edu.mk;
phone number: +38923235411
page: 5
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RESULTS
Clinical characteristics and treatment response
All clinical data of the patients included in the study
are summarized in Figure 1. The median age at diagnosis
was 4 years (range: 0-11). The white blood cell (WBC)
count at diagnosis was higher than 20 x 10^9/L in 34%
of the patients, while CNS infiltration was a less frequent
event (10%). The percentage of patients with poor pred-
nisolone response (PPR; absolute blast count >1000 in pe-ripheral blood on day 8) was 13%. Following the ALL-IC-
BFM 2002 classification [19], 15% of the patients (8/52)
were stratified into the high-risk group. One patient (aged
<1 year) was enrolled in the Interfant-06 trial, while two
patients moved out of the country after diagnosis and were
lost for follow-up. The remaining patients received treat-
ment according to the intermediate-risk protocol. NGS-
MRD data for two time-points from therapy onset (day 33
and day 78) were available for 43 patients. A positive MRD
status (MRD ≥ 1 x 10^(-4)) was detected in 24/43 (56%)
and 7/43 (16%) of the patients on days 33 and 78, respec-
tively [17]. During a median follow-up of 46 months, five
patients relapsed. All relapses occurred between 17 and
32 months after diagnosis.
Molecular alterations at diagnosis
The most common genetics subtype in the study pop-
ulation was high-hyperdiploidy, present in 33% (18/55) of
the patients, followed by the ETV6::RUNX1 subtype in
15% (8/55). Four other patients (7%) were identified with
the TCF3::PBX1 subtype, and one patient each (1,8%)
with the MLL::AFF1, BCR::ABL1 and iAMP21 subtypes.
At diagnosis, at least one copy number alteration in the
selected genes was detected in 88% (44/50) of the patients,
with 75% of them showing more than two alterations (Fig-
ure 1). The most common alteration was the duplication of
the PAR1 region (CRLF2, CSF2RA, IL3RA), while PAR1
deletion was observed in one patient. Deletions in the
IKZF1 gene were present in 10% (5/50) of the patients,
in two of which partial, and none of them presented with
the IKZF1plus profile [20]. CDKN2A/2B gene deletion was
detected in 28% (14/50) of the patients (8 heterozygous, 6
homozygous), while PAX5 gene deletion in 22% (11/50),
co-occurring in six patients. Deletion and duplication of the
ETV6 gene was detected in 6 (12%) and 2 (4%) patients,
respectively. BTG1 gene deletion was observed in three
patients (one of them homozygous), while duplication was
found in two other patients. RB1 gene deletion was present
in three patients, while the EBF1 gene was deleted in one
patient and duplicated in another. Deletion of TP53 gene
was present in two patients (4%).
CNAs and SNVs in the matched
diagnosis-relapse samples
Clinically relevant alterations in the five patients with
relapse (No. 4, 10, 32, 35 and 36 from Figure 1), including
the findings from the WES analysis, are summarized in
Table 1. Among them, two patients were identified with
high-hyperdiploidy, one of which had an additional EBF1
gene duplication; one patient had a BCR::ABL1 hybrid
transcript along with CDKN2A/2B and PAX5 gene partial
deletions, and one patient was found to have TP53, EBF1,
and ETV6 gene deletions at diagnosis. The aneuploidy,
hybrid transcript, and the EBF1 and ETV6 gene altera-
tions were preserved in the matched relapse samples. The
deletion of TP53 was present in both diagnosis and relapse
samples in one patient and gained at relapse in another. By
contrast, the CDKN2A/2B deletions were either gained or
lost in the relapse samples, and PAX5 gene deletion was
lost in the relapse sample. Additionally, single nucleo-
tide variants in three genes were involved in the relapse
samples, including the epigenetic regulator CREBBP, the
RAS pathway gene NRAS, and the DNA mismatch repair
gene MSH2. Notably, alterations in these genes were not
observed in the five diagnostic samples from patients in
remission that were analyzed by WES. Mutations affect-
ing the histone acetyltransferase (HAT) domain of the
CREBBP gene were persistent in the diagnostic and re-lapse clones in a single patient, acquired at relapse in two
patients or involved a change in the same amino acid in
this domain in one patient (from R1446H in the diagnostic
clone to R1446C in the relapse clone). The p.G12D muta-
tion in the NRAS gene was the second most common altera-
tion in these patients, either gained or lost in the relapse
samples of two patients, while the mutation in MSH2 was
present in the relapse sample only, of one patient. Addition-
ally, a mutation in FLT3 was observed in the diagnostic
clone in one patient who relapsed, but mutations in this
gene were also found in 2 out of 5 patients in remission.
The type of SNV for each gene is presented in Table S1.
Clonal evolution of the relapses
Fragment analysis and Sanger sequencing of the im-
munoglobulin heavy chain genes in the matched diagnosis
and relapse samples revealed the presence of the same
clonally rearranged sequences at relapse as those observed
at diagnosis in four patients (No. 4, 10, 35 and 36) (Table
S2). In two of these patients (No. 10 and 36), one of the
clonal rearrangements was lost at relapse. Nevertheless,
in these two patients, we observed the same genetic al-
terations in both the diagnosis and relapse samples, with
additional gain of TP53 deletion in one of them. In the
other two patients with the same clonal rearrangements, we
observed gains (in CDKN2A/2B, CREBBP, and NRAS) and
losses (in FLT3 and NRAS) of genetic alterations at relapse.
The last patient (No. 32) retained only the BCR::ABL1
hybrid transcript at relapse, lost the CDKN2A/2B and PAX1
deletions, and gained alterations in CREBBP and MSH2.
This patient also showed different clonally rearranged se-
quences at relapse, presenting three rather than five clonal
sequences detected at diagnosis, of which only one was
same in both samples (Table S2).
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