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Received: 29 October 2020    Revised: 31 December 2020    Accepted: 12 January 2021

DOI: 10.1111/ijlh.13477

ORIGINAL ARTICLE

Epigenetic analysis reveals significant differential expression of

miR-­378C and miR-­128-­2-­5p in a cohort of relapsed pediatric
B-­acute lymphoblastic leukemia cases

Prateek Bhatia1  | Minu Singh1 | Aditya Singh1 | Pankaj Sharma1 | Amita Trehan1 |

Neelam Varma2

1
Department of Pediatrics, Pediatric
Hematology Oncology Unit, Postgraduate
Institute of Medical Education & Research,
Chandigarh, India
2 oresistance and relapse in pediatric ALL, and hence in current pilot study, we tried to
Department of Hematology, Postgraduate
Institute of Medical Education & Research,
Chandigarh, India
Correspondence
Prateek Bhatia, Pediatric Hematopathology,
Pediatric Hematology-­Oncology Unit,
Advanced Pediatric Centre, PGIMER,
160012 Chandigarh, India.
Email: prateekbhatia@rediffmail.com
Funding information
Major funding for the study was received
from Department of Science-­Science
Engineering and Research Board (DST-­
SERB; Sanction No. EMR/2017/000104),
New Delhi and partial funding support from
Intramural PGIMER, Chandigarh Grant.
Abstract
Introduction and Objective: Epigenetic changes play a major role in mediating chem-
identify DNA methylation, miRNA expression, and copy number variations (CNVs) in
a cohort of relapse pediatric B-­ALL cases.
Methodology: DNA methylation, miRNA expression, and CNV analysis were per-
formed in a total of 14, 16, and 18 cases as diagnosis-­relapse samples. Briefly, DNA
methylation was performed using Infinium HumanMethylation850 chip and data an-
alyzed using RnBeads. miRNA was sequenced on illumina NextSeq500 platform for
20M 75bp SE reads and analyzed by DESeq2. CNVs were assessed by MLPA assay
using the ALL P-­335 probemix kit and analyzed by coffalyzer.net.
Results: On methylation analysis, oncogenes MYCN, MYB, and EGFR and tumor sup-
pressor genes MDM4 & BCL11B were found differentially expressed as compared to
controls (p-­0.03). In addition, protooncogenes—­A XL, HCK, MED12, and ETS2—­were
hypomethylated/overexpressed in 4 or more cases (P < .05). miRNA analysis revealed
significant differential expression of miR-­128-­2-­5p and miR-­378C (p-­4.4e-­15 and p-­
6.4E-­12) in relapse samples. CNV analysis revealed that frequency of good and inter-
mediate/poor risk CNV profile at diagnosis was nearly equal (40% vs 60%). However,
CDKN2A/2B and IKZF1 gene CNVs if present in initial diagnostic clone usually per-
sisted in relapse clone.
Discussion and Conclusion: Our pilot study highlights two miRNAs (miR-­128-­2-­5p
and miR-­378C) as possible candidate biomarkers of relapsed B-­ALL. However, these
miRNAs and hypomethylated protooncogene signature noted in our data needs vali-
dation in a larger series of B-­ALL.

KEYWORDS

B-­ALL, CNVs, methylation, miRNA

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1016     © 2021 John Wiley & Sons Ltd wileyonlinelibrary.com/journal/ijlh Int J Lab Hematol. 2021;43:1016–1023.

BHATIA et al.
1 |  I NTRO D U C TI O N
Acute lymphoblastic leukemia is one of the most common childhood
hematological malignancies.1,2 At our centre in Northern India, we
see approximately 130-­150 newly diagnosed ALL per year, while
elsewhere in India, near about 10 000 to 12 000 new cases of pe-
diatric ALL occur per year.3 Despite vast improvement in the treat-
ment of ALL, 15%-­20% of leukemia cases relapse and account for
more deaths from cancer in children than any other malignancies.4
Therefore, relapse remains a major challenge to clinicians in the suc-
cessful treatment of children with ALL.
Epigenetics is the study of phenotypic changes in a cell that are
not inherited through cell division. These changes at genomic level
can be identified as DNA methylation patterns of promoter genes
or CpG islands and by miRNA-­mediated control of transcriptional
activity. Genome-­wide DNA methylation studies have been used to
identify distinct biologic subtype and predict treatment outcome of
5-­8
myeloid leukemia and acute lymphoblastic leukemia. DNA meth-
ylation changes play a major role in mediating chemoresistance and
relapse in pediatric ALL.9 Promoter hypermethylation leading to tran-
scriptional silencing is also reported in pediatric relapse ALL. During
recent years, miRNAs have emerged as major players in the complex
networks of gene regulation and have been implicated in various as-
pects of human disease. Over the past 10 years, the emerging role of
miRNAs in mediating chemoresistance and relapse of acute lympho-
blastic leukemia is under intense investigation. Aberrant expression
of miRNA has been detected in ALL, and some of the studies have
correlated this with respect to T-­ALL, MLL-­rearranged, TEL-­AML1,
E2A-­PBX, BCR-­ABL, and pre B-­ALL with hyperdiploidy.10,11 Earlier
studies reported that miRNAs are not only involved in the regulation
of gene expression, but also involved in leukemogenesis including
pediatric acute lymphoblastic leukemia.12,13 Schotte et al showed
that miRNA gene expression is deregulated in acute lymphoblastic
leukemia with the most significantly deregulated miRNAs being miR-­
125b, miR-­221, miR-­222, and miR-­155.11-­13
In current pilot study, we hypothesized that a detailed analysis of
DNA methylation, miRNA expression, and copy number gene vari-
ations (CNVs) could help in identifying novel biomarkers of relapse
ALL in our cohort. Moreover, such integrated epigenetic studies are
lacking from our sub-­continent considering a geographically and
ethnically distinct population.
2 |  M ATE R I A L S A N D M E TH O DS
A total of 14, 16, and 18 diagnosis (D0)-­disease relapse (DR) sam-
ples were enrolled for DNA methylation, miRNA, and CNV studies,
respectively. Only 2 paired samples were derived from two differ-
ent patients in miRNA analysis, while the rest patient paired sam-
ples were common to all three analysis. A written informed consent
was taken from patients/guardians for sample collection, and the
study was duly approved by the Institute's Ethics Committee and
the Departmental review board.
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      1017
Of 2-­3  mL EDTA blood sample was withdrawn from patients
at diagnosis and disease relapse. All cases had >90% blasts in pe-
ripheral blood. RNA and DNA were then extracted separately from
PBMCs from each sample and stored at −80c after noting quantity
and quality by Nano quant and qubit assessment. All pediatric leu-
kemia cases at our institute undergo flow cytometry evaluation at
diagnosis with a standard panel of markers to identify B-­/ T-­cell lin-
eage and are also routinely worked up for recurrent translocations
and or hyperdiploidy by both FISH and RT-­PCR. Treatment evalua-
tion was assessed by doing D8 absolute blast count, Day 35 bone
marrow for blast persistence and Day 35/end of induction minimal
residual disease assessment (MRD; cutoff 0.01%) by flow cytometry.
All patients were treated under ICicLe (Indian Childhood collabora-
tive leukemia protocol) treatment protocol which is a modification
and adaptation of UK MRC 2003 protocol. This protocol is based
on an initial seven-­day prednisolone prephase and thereafter 3 or
4 drug-­based induction chemotherapy (vincristine, prednisolone
L-­asparaginase, and daunorubicin) depending upon initial NCI risk
category and genetic analysis results. In addition, the protocol ran-
domizes all cases to mitoxantrone in place of doxorubicin during de-
layed intensification phase.
2.1 | DNA methylation methodology
A total of 14 DNA samples from 7 ALL patients at diagnosis (day
0) and their paired sample at relapse (Day-­R) along with 2 con-
trols were selected for methylation studies. Bisulfite conversion
of 200-­500  ng gDNA was performed prior to the initiation of the
assay using EZ DNA methylation-­Gold kit (Cat No. D5005; Zymo
Research). The bisulfite-­treated ssDNA was then quantified using
the Eukaryotic Total RNA setting with an RNA pico-­chip (Cat No.
5067-­1513; Agilent Technologies) on Agilent 2100 Bioanalyzer.
The DNA methylation assay was then performed using the human
Infinium MethylationEPIC 16 sample BeadChip (Cat No. WG-­
317-­1001; Illumina) with 850 000 genome-­wide methylation sites on
illumina iscan scanner as per Infinium HD methylation assay guide.
After run as per protocol, DNA methylation values, described as
beta values (continuous variables between 0 and 1, representing the
ratio of the intensity of the methylated bead type to the combined
locus intensity) were recorded for each locus in each sample via
GenomeStudio software. Complete analysis was performed using
RnBeads software.
2.2 | miRNA analysis by NGS
A total of 16 RNA samples from 8 ALL patients at diagnosis (day 0)
and their paired sample at relapse (Day-­R) were enrolled for miRNA
analysis. RNA was extracted using QIAamp Blood RNA kit (Cat No.
52304; Qiagen) same day. The RNA was stored at −80˚C till fur-
ther use. Quality check of RNA was performed before sequencing
using both Qubit RNA HS assay kit (Cat No. Q32852; ThermoFisher
 |
1018       BHATIA et al.
Scientific) on Qubit Fluorometer and on Bioanalyzer using Agilent
small RNA kit (Cat No. 5067-­1548; Agilent Technologies) for RIN
value calculation. NEBNext Multiplex Small RNA Library Prep kit
(Cat No. E7580S; New England BioLabs Inc.) was used for prepara-
tion the libraries for miRNA sequencing. The libraries prepared were
then size selected on a gel to yield size-­specific (140 bp to 160 bp)
small RNA library. They were sequenced on Illumina NextSeq500
for 20 M 75 bp SE reads. The FastQC and Cutadapt (ver 1.16) were
used for quality check and preprocessing of the reads. Structural
RNA contamination, including ribosomal RNAs and transfer RNA
sequences, were removed using Bowtie2. Reads were mapped to
the known miRBase mature and precursor using miRDeep2 and
the Randfold (ver-­2.0) P-­values were calculated for the subset of
reads that mapped to corresponding miRNA and its precursor using
Randfold -­ S (S represents sequence). MiRDeep2 was also used to
predict presence of novel miRNA. The identified Novel miRNA was
filtered for Randfold filtration of YES and Star read count greater
than or equal to 1. The Target predictions for Novel miRNA were
performed using Miranda. Differential miRNA analysis between di-
agnosis samples (Group-­1) and paired relapse samples (Group-­2) was
performed using DESeq2.
2.3 | Copy number Variations by MLPA
MLPA analysis for CNVs was performed in a total 18 DNA samples
from 9 ALL patients at diagnosis (day 0) and their paired sample at
relapse (Day-­R). Three controls were also run for data normalization.
DNA extracted was run using the SALSA MLPA P-­335 ALL Probemix
(Cat No. P335-­025R; MRC Holland Netherlands) as per manufac-
turer's instructions on ABI 3500 capillary sequencer, and data were
analyzed using coffalyzer.net software. The scoring of copy num-
ber abnormalities was done on the basis of dosage quotient (DQ)
as follows: normal copy number (0.80-­1.20); heterozygous deletion
(0.40-­0.65); homozygous deletion (0.00); heterozygous duplication
(1.3-­1.65); and homozygous duplication (1.75-­2.15).
3 |   R E S U LT S
3.1 | Demographic, clinical, hematological, and
outcome data of all enrolled cases
A total of 12 pediatric (age  ≤  12  years) relapse B-­ALL cases were
enrolled during August 2015 to May 2019, of which 9 cases were
paired as their diagnostic baseline DNA and RNA samples were
available in our biobank. The median age of patients was 5  years,
and male to female ratio was 11:1. The median of WBC count at
the time of diagnosis was 50 000/µL. According to National Cancer
Institute (NCI) disease stratification, 6 patients were categorized
as low risk (WBC  <  50  000; age  <  10  years) and 6 were high risk
(WBC  ≥  50  000/µ; age  ≥  10  years). Upon molecular and cytoge-
netic testing, two patients were positive for ETV6-­RUNX1, one for
1751553x, 2021, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ijlh.13477 by The Director Pgimer, Wiley Online Library on [11/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
E2A-­PBX and one had high hyperdiploidy. The relapse samples of
these cases harbored the same primary diagnostic molecular and
ploidy change.
As per the treatment protocol followed at our institute, minimal
residual disease (MRD) was tested at the day 35 of the induction
phase. Out of 12 B-­ALL patients, MRD data were available for 10
patients. Four out of 10 patients had high (>0.01%) MRD at day 35.
The relapse data were available for 8 patients, of which, 4 had medi-
astinal relapse, 3 had isolated testes relapse and 2 had isolated CNS
relapse. During the follow-­up of relapse cases, 8 patients opted for
therapy while 3 left treatment and 2 patients died following relapse.
3.2 | DNA methylation results
A total 8 comparisons between 16 samples were performed (in-
cluding two control samples). These included 1 global comparison
between all samples and 7 paired analysis between D0 and DR for
each sample. RnBeads was used to perform the exploratory and
differential methylation analysis and methylation level were repre-
sented as beta values. Beta values greater than 0.70 were regarded
as hypermethylated and less than 0.3 as hypomethylated. The analy-
sis included both CpG sites and genomic regions (tiling, promoters,
genes, and CpG islands). During quality preprocessing of data, a total
of 20 511 probes and 17 371 sites were filtered out based on specific
criteria of unreliable measurements (probe beta value corresponding
P-­value was above threshold) and overlap with SNPs, respectively.
Hence, finally 846  384 probes for 14 samples were retained for
analysis. The methylation β values were normalized using the BMIQ
normalization method.
The global (all samples together) as well as single paired genome-­
wide methylation comparison between diagnostic and relapse
samples did not reveal any statistically significant expression pro-
file among tumor suppressor, oncogene, and cell-­cycle genes (See
Supplementary Figure  S1 and Figure  1A,B). Moreover, distribution
of methylation sites according to different region-­wise analysis too
did not reveal significant difference among diagnostic and relapse
samples (see Figure  1C,D). However, significant difference was
noted between differential methylation pattern of control samples
and leukemia samples with oncogenes like MYCN, MYB, and EGFR
and tumor suppressor genes like MDM4 and BCL11B and cell-­cycle
genes like HSPA2, PSMC5, NEK7, and CEP250 being differentially
methylated (P-­.03) (See Supplementary Figure S1 and Figure 1A,B).
A total of 427 175 (50.6%) probes had higher methylation at diagno-
sis, and 416 225 (49.4%) had higher methylation at relapse. Of these,
8 tumor suppressor genes were hypermethylated in relapse sam-
ples. In addition, 9 tumor suppressor genes were hypomethylated
and 45 unaltered in relapse samples. Among oncogenes, 46 were
hypomethylated (including 3 miRNA coding genes), 38 were hyper-
methylated, and 74 were unaltered at relapse (see Supplementary
Table  S1). On further analysis, it was noted that protooncogenes
AXL, HCK, MED12, and ETS2 were hypomethylated (possibly overex-
pressed) in 4 or more cases (P < .05).

BHATIA et al.
(A)
(C)
F I G U R E 1   A, Heat map for beta values of all samples (n = 14) and controls (n = 2) with respect to tumor suppressor genes. B, Heat map
for beta values of all samples (n = 14) and controls (n = 2) with respect to oncogenes. C, Distribution of methylation sites in promoter region
for all cases at diagnosis and at relapse. D, The distribution of number of CpG sites for all cases at diagnosis and at relapse
3.3 | miRNA expression results
A total of 16 samples (8 paired diagnosis-­relapse) were sequenced
for small/microRNA. The data generated for each sample were
greater than 0.14 GB. After adapter trimming, the read length dis-
tribution indicated that majority of the reads had an average length
of 22. The alignment percentage to the reference genome (hg19)
was around 68 to 97%. The alignment percentage to the microRNA
was around 4 to 71%, respectively. The uncontaminated reads were
used for known and novel microRNA analysis using mirDeep2 tool.
The data generated are summarized in Table 1. Total number of miR-
NAs identified in each sample ranged from 126 to 389. Of these,
the known miRNA ranged from 122 to 322 and novel miRNA from
3 to 101 (Table 2). miR-­92a-­1-­5p was the most predominant miRNA
found across all the 16 samples. The hairpin structures of novel
miRNA predicted in each sample are presented in Supplementary
data file. A total 8 pairwise comparisons were performed. The
ratio of read counts was taken as the fold change. Those miRNAs
which were found to be 2 standard deviation away from the mean
(mean  + 2 ×  standard deviation, mean  -­  2  ×  standard deviation)
were considered statistically significant. The result details on pair-
wise comparison are highlighted in Table 1. In addition, a cumulative
grouped sample (diagnosis/Group-­1 vs relapse/Group-­2) differential
expression analysis was also performed using DEseq2. Significant
miRNAs were filtered based on P-­value  <  0.05, and those with
Log2foldchange ≥1 and ≤1 were taken as upregulated and down-
regulated miRNA, respectively. A total of 16 miRNA were noted to
be differentially expressed (Log2fold change ≥1 or ≤1) between the
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      1019
(B)
(D)
diagnostic and relapse group. Of these, 7 were upregulated and 9
downregulated (Figure 2A). However, only two miRNAs—­miR-­378c
and miR-­128-­2-­5p, out of 7 that were upregulated, were significantly
differentially expressed with adjusted P-­value < .05 (p-­4.4e-­15 and
p-­6.4E-­12, respectively) and >10-­fold upregulation as compared to
other miRNAs at relapse (refer volcano plot Figure  2B). The Heat
map in Figure 2A shows that these two miRNAs were upregulated
in 3 samples each.
3.4 | Copy number analysis results
The .fas files obtained after capillary electrophoresis in 18 samples
(9 paired diagnosis-­relapse samples) were analyzed using coffalyzer.
net software. The data were aligned with the reference control sam-
ple as well as with internal reference probes and the copy number
variations were noted in IKZF1, PAX5, EBF1, BTG1, RB1, CDKN2A/B,
PAR1, CSF2RA, IL3RA, and CRLF2 genes based on their DQ values.
CNVs good risk (GR) or intermediate/poor risk (I/PR) was de-
fined according to the report by Moorman et al14 On analysis of
MLPA data, copy number abnormalities (CNVs) were noted in total
of 12/18 (67%) samples. Deletions were more common (12/18)
as compared to duplications in the genes (4/18). Most common
deletions noted were that of CDKN2A/2B in 5 samples each, fol-
lowed by that of RB1, IKZF1, BTG-­1, and PAX-­5 in 3 samples each.
Duplications were primarily seen in CRLF-­2 (3 samples at diagnosis
and 2 at relapse) and ETV6 and PAX-­5 gene (2 samples each), fol-
lowed by duplication of EBF1 and IKZF1 in one sample each. None
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1020       BHATIA et al.

TA B L E 1   Data summary for miRNA run by NGS

Mean read Total data Mean read quality Known Novel Total
Sample Group Sample ID Total reads length (bp) in Gb (Phred score) GC% miRNA miRNA miRNA

Diagnosis S-­1_SK 26 327 002 51 1.343 38.42 50.93 322 46 368

samples group S-­2_FS 2 723 842 51 0.139 37.58 54.71 122 4 126
S-­3_AR 22 574 361 51 1.151 38.05 55.55 200 15 215
S-­4_AM 26 742 082 51 1.364 38.36 53.98 264 17 281
S-­5_RU 22 048 721 51 1.124 38.48 53.8 311 45 356
S-­6_AN 7 484 840 51 0.382 37.96 54.27 246 10 256
S-­7_YO 2 906 244 51 0.148 37.41 55.27 144 3 147
S-­8 _NK 27 112 660 51 1.383 37.16 50.37 162 6 168
Paired relapse S-­10_FSR 49 017 394 51 2.500 37.48 54.41 288 101 389
samples group S-­11_ARR 14 081 167 51 0.718 37.45 52.49 174 16 190
S-­13_RUR 20 939 538 51 1.068 37.4 53.11 277 38 315
S-­14_ANR 26 807 018 51 1.367 37.42 53.04 286 41 327
S-­15_YO 18 744 394 51 0.956 37.25 53.67 205 10 215
S-­16_NK 24 409 740 51 1.245 37.49 52.3 177 8 185
S-­9_SKR 19 023 305 51 0.970 37.52 51.22 307 30 337
S12_AMR 5 669 821 51 0.289 37.7 51.32 302 35 337

of the case with duplications were noted to have hyperdiploidy on
cytogenetic analysis. On classification of abnormalities into good
and intermediate/poor risk groups, a total of 10 samples (5 diag-
nostic and 5 relapse) showed intermediate/poor risk CNVs while 4
diagnostic and 4 relapse samples had good risk CNV profile. The
detailed CNV profile predominantly involving deletions in diag-
nostic and relapse samples is highlighted in Figure 3 as a heat map.
4 |  D I S CU S S I O N
In current study, first of its kind from our sub-­continent, we per-
formed epigenetic analysis of pediatric paired diagnosis-­relapse
B-­ALL cases targeting DNA methylation status and miRNA expres-
sion events. In addition, we did paired analysis for copy number
variations. The study though performed in a small cohort is relevant,
considering the data that epigenetic events play an important part
in ALL relapse and detection of a biomarker might help prevent or
detect early-­onset disease relapse thereby substantially reducing
overall morbidity and mortality.
Methylation studies performed using 850K Infinium BeadChip
in our cohort revealed 5 protooncogenes (AXL, HCK, MED12, and
ETS2) to be hypomethylated and possibly overexpressed in 4 or more
patients (P < .05). AXL, a member of TAM family of receptor tyrosine
kinases, when overexpressed, is well-­known to provide a chemopro-
tective niche to acute myeloid leukemia cells by enhancing expres-
sion of its ligand, Gas6, by tumor-­infiltrating myeloid cells, thereby
increasing risk of relapse.15 Recent evidence in a study by Park
et al also revealed that AXL phosphorylation activates Flt3 in AML
cells and its inhibition reduced cellular proliferation.16 HCK promoter
has been shown to have aberrant methylation status in different he-
matological malignancies. Hoshino et al showed that HCK acts as a
protooncogene and is overexpressed/hypomethylated in CML and
Ph-­positive ALL while it acts as a tumor suppressor gene and signi-
fies poor prognosis if hypermethylated in Ph-­negative ALL.17 MED12
is a member of mediator complex and has important epigenetic func-
tion in that it controls transcriptional activity of various genes by
acting as a cross-­t alk protein between RNA Pol II and transcription
factors.18 Cancer cells exploit this mechanism role of MED12 and
hence lead to aberrant expression of many genes including other im-
portant protooncogenes related to tumor development and progres-
sion. ETS2 is a transcription factor protooncogene that is involved in
regulation of cellular proliferation and differentiation, and its over-
expression has been linked to complex karyotype in acute myeloid
leukemia (AML) and in development of Down Syndrome associated
AMKL (acute megakaryocytic leukemia).19,20 Though all above pro-
tooncogenes have been implicated in cancer development and pro-
gression, their current status and role in relapse ALL has not been
much explored and it needs to be further assessed and analyzed in
larger prospective studies.
Our study also yielded important information on significant differ-
ential overexpression of two miRNAs (miR-­378c and miR-­128-­2-­5p; >10
log fold) on cumulative diagnosis (Group-­1) vs paired relapse (Group-­2)
sample analysis using DESeq2. Han et al in 2011 found significant dif-
ferential expression of miR-­128b in relapse pediatric ALL samples and
suggested that this miRNA could possibly control a tumor suppressor
protein, and hence, its overexpression leads to relapse in ALL, though
they could not identify the target.10 miR-­128b has also been identified
as a differentially expressed miRNA in many tumor cells, though its role
as a tumor regulator is controversial as few studies show it to act as a
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BHATIA et al.       1021

TA B L E 2   Differentially expressed
Comparison (Patient Upregulated Downregulated
miRNA summary in paired comparison
sample ID) miRNA miRNA Log2 foldchange cutoff

S-­1_SK vs S-­9_SKR 9 3 UP miRNAs > 2.39 & Down

miRNAs < −3.49
S-­2_FS vs S-­10_FSR 2 4 UP miRNAs > 12.06 & Down
miRNAs < 2.00
S-­3_AR vs S-­11_ARR 3 1 UP miRNAs > 2.93 & Down
miRNAs < −2.53
S-­4_AM vs S12_AMR 3 7 UP miRNAs > 3.85 & Down
miRNAs < −1.94
S-­5_RU vs S-­13_RUR 5 3 UP miRNAs > 1.86 & Down
miRNAs < −4.20
S-­6_AN vs S-­14_ANR 6 5 UP miRNAs > 5.66 & Down
miRNAs < −1.30
S-­7_YO vs S-­15_YO 2 4 UP miRNAs > 4.07 & Down
miRNAs < −0.11
S-­8 _NK vs S-­16_NK 3 1 UP miRNAs > 3.82 & Down
miRNAs < −2.97

F I G U R E 2   A, Heat map for the 16 most significantly differentially expressed miRNAs (upregulated and downregulated based on P-­
value < .05 and Log2foldchange ≥1 and ≤1, respectively) in Diagnosis Vs Relapse ALL samples. B, Volcano plot showing >10 fold change in
miR-­378c and miR-­128-­2-­5p expression in Diagnosis vs Relapse ALL comparison

tumor suppressor while others show it to be oncogenic.21-­26 However,
many recent studies in acute leukemia cases have shown that miR-­
128b is significantly overexpressed in pediatric ALL cases at diagnosis
27-­30
than controls and in ALL cases more than in AML cases. In addi-
tion, in vitro studies performed have shown miR-­128b to be involved
in development of drug resistance to vincristine and prednisolone,
pointing toward a possible mechanism for disease relapse in ALL.31
miR-­378c is a tumor suppressive miRNA and studies have shown it to
be downregulated in cancers especially bladder and colon cancers and
its down-­regulation leads to tumor metastasis and poor outcome.32,33
However, it is difficult to explain the current relation of overexpression
of this tumor miRNA in pediatric ALL relapse samples and hence needs
to be studied functionally in detail in a larger cohort involving ALL re-
lapse cases. The significant overexpression of both these miRNAs in
our study, especially miR-­128b, suggests that they have the potential
to be employed as biomarkers for detecting early relapse in pediatric
ALL. miRNAs are continuously being secreted by malignant/leukemic
cell clones into extracellular fluid and tissue spaces as components of
extracellular vesicles. Moreover, miRNAs are quite stable as compared
to mRNA and hence have potential to be detected by serial monitoring
of platelet-­poor plasma derived from peripheral blood samples during
different time intervals in ALL cases undergoing treatment (baseline,
 |
1022       BHATIA et al.
end of induction, mid-­consolidation and during mid/end maintenance
and posttherapy completion every 3-­6 months for 3 years). The log-­
change in expression of these miRNAs can be monitored by RQ-­PCR
and or digital PCR and any significant log-­fold change detected at
above time points would signify either poor response to therapy (if at
end induction or mid-­consolidation) or clue to a possible disease re-
lapse in near future (if at mid/end maintenance and post therapy com-
pletion at any point).
Other than the epigenetic analysis, we also performed copy num-
ber variation (CNV) analysis in paired diagnosis-­relapse ALL samples.
We analyzed the results as per the mentioned cutoffs for DQ in meth-
odology section since all of our samples had >90% blasts in peripheral
blood at time of sampling. However, one needs to remember that the
cutoffs may vary as per blast % in different settings, within the range of
test sensitivity. Our analysis of MLPA data revealed that CDKN2A/2B
deletional events are most common CNVs in pediatric ALL. Moreover,
CDNAK2A/2B and IKZF1 deletions if present in the diagnostic clone
usually persist in the relapse clone too, suggesting their important role
in propagating the development of leukemia as secondary genetic ab-
normalities in the two-­hit leukemia development model.
5 |  CO N C LU S I O N
The current pilot study sheds light on the epigenetic findings in re-
lapse pediatric ALL and highlights that miR-­378c and miR-­128-­2-­5p
have potential to be used as biomarkers for early detection of re-
lapse ALL since their expression levels were >10log fold higher in
relapse samples. In addition, the study points to possible role of hy-
pomethylated protooncogenes like AXL in disease relapse and sug-
gests prospective larger cohort studies to delineate the pathways
associated with these genes so that targeted treatments can be ex-
plored to treat relapse cases.
1751553x, 2021, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ijlh.13477 by The Director Pgimer, Wiley Online Library on [11/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
F I G U R E 3   Heat map of copy number
variations in 9 paired B-­ALL samples at
diagnosis and relapse
AC K N OW L E D G E M E N T S
We wish to thank DST-­SERB, New Delhi and PGIMER, Chandigarh
for funding support for the current study.
C O N FL I C T S O F I N T E R E S T
None to declare.
AU T H O R C O N T R I B U T I O N S
Prateek Bhatia involved in conceptualization and writing-­original
draft preparation. Minu Singh involved in methodology and data
curation. Aditya Singh and Pankaj Sharma involved in software and
investigation. Amita Trehan and Neelam Varma involved in review-
ing and editing.
DATA AVA I L A B I L I T Y S TAT E M E N T
The data related to the study are available as a single Supplementary
data file.
ORCID
Prateek Bhatia  https://orcid.org/0000-0001-6096-3587
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S U P P O R T I N G I N FO R M AT I O N
Additional Supporting Information may be found online in the
Supporting Information section.
How to cite this article: Bhatia P, Singh M, Singh A, Sharma P,
Trehan A, Varma N. Epigenetic analysis reveals significant
differential expression of miR-­378C and miR-­128-­2-­5p in a
cohort of relapsed pediatric B-­acute lymphoblastic leukemia
cases. Int J Lab Hematol. 2021;43:1016–­1023. https://doi.
org/10.1111/ijlh.13477