MIR4435-2HG as a Novel Predictive Biomarker of Chemotherapy Response and Death in Pediatric B-cell All

doi:10.21203/rs.3.rs-3486950/v1

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Research Article

MIR4435-2HG as a Novel Predictive Biomarker of Chemotherapy Response and Death in Pediatric B-cell All

https://doi.org/10.21203/rs.3.rs-3486950/v1

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Background

Although B-cell acute lymphoblastic leukemia (B-cell ALL) survival rates have improved in recent years, Hispanic children continue to have poorer survival rates. There are few tools available to identify from the time of diagnosis whether the patient will respond to induction therapy. Our objective was to identify predictive biomarkers of treatment response, which could also serve as prognostic biomarkers of relapse and death, by identifying methylated and differentially expressed genes between patients with positive minimal residual disease (MRD+) and negative minimal residual disease (MRD-).

Methods

Tumor blasts were separated by immunomagnetic column and subsequently DNA and RNA were extracted. DNA methylation and mRNA sequencing assays were performed on 19 bone marrows from Hispanic children with B-cell ALL. Partek Flow was used for transcript mapping and quantification, followed by differential expression analysis using DEseq2. DNA methylation analyses were performed with Partek Genomic Suite and Genome Studio. Gene expression and differential methylation were compared between patients with MRD- and MRD + at day 15 and at the end of induction chemotherapy. Overexpressed and hypomethylated genes were selected and validated by RT-qPCR in samples of validation cohort. The predictive ability of the genes was assessed by logistic regression. Survival and Cox regression analyses were performed to determine the association of genes with death. The association of genes with relapse was assessed by RT-qPCR in relapsed patient samples and validated using TARGET-PANCER data.

Results

DAPK1, CNKSR3, MIR4435-HG2, CTHRC1, NPDC1, SLC45A3, ITGA6, and ASCL2 were overexpressed and hypomethylated in MRD + patients. The overexpression of DAPK1, ASCL2, SCL45A3, NPDC1 and ITGA6 can predict non-response at day 15 and refractoriness. Additionally, higher expression of MIR4435-2HG increases the probability of non-response, death, and the risk of death. MIR4435-2HG is also overexpressed in relapse samples. Finally, MIR4435-2HG overexpression, together with MRD+, are associated with poorer survival, and together with overexpression of DAPK1 and ASCL2, it could improve the risk classification of patients with normal karyotype.

Conclusions

MIR4435-2HG is a potential predictive and prognosis biomarker in children with B-cell ALL.

B-cell acute lymphoblastic leukemia

biomarkers

MRD

gene expression

DNA methylation

prognosis

treatment response

B-cell acute lymphoblastic leukemias (B-cell ALL) are the most frequent neoplasms in children (1). Cure rates for acute lymphoblastic leukemias (ALL) have improved remarkably in the last four decades; nevertheless, while developed countries achieve 80%, cure rates in developing countries are around 60% [2]. The mechanisms underlying these differences in survival rates are still unknown; however, some studies have shown that Hispanic children have worse survival and treatment response compared to White and Asian children, even using the same treatment protocols (3–5).

Currently, clinical parameters such as leukocyte count, age, extramedullary infiltration, chromosomal translocations, and minimal/measurable residual disease (MRD) classify patients into risk groups. MRD is the most used variable to define treatment response (6, 7). However, due to low survival in our patients, it is possible to propose that those variables do not fully define risk groups, which leads to incorrect selection of chemotherapy protocol, affecting patient survival (8).

In ALL, gene expression alterations not only result from mutations; alterations at the epigenetic level also play a relevant role in this pathology (9–12). Thus, epigenetic alterations, including aberrant DNA methylation, could act as important molecular mechanisms in developing resistance to treatment of ALL (12). In bone marrow (BM), DNA methylation patterns change during normal hematopoiesis and play an essential role in lineage differentiation (13, 14). As in normal cells, tumor cells may also depend on specific DNA methylation patterns to acquire their phenotype and maturation patterns (13, 15, 16). Therefore, the characterization of aberrant patterns in DNA methylation in tumors can provide important clues about how gene expression is regulated in these pathologies (11). Hogan et al, found that patients with relapses presented promoter hypermethylation and identified a clear signature of differentially expressed genes at the time of diagnosis and relapse; moreover, this signature differs between early-relapse patients compared to late-relapse patients (17). Although differential methylation and gene expression patterns have been observed between samples at diagnosis and in relapse, it should be determined whether these variables could be tools to predict treatment response, including relapse or death. Also, a CpG island methylation analysis identified candidate genes as biomarkers of pediatric ALL subgroups and their correlation with disease prognosis (18). Identifying genomic markers, derived from methylation and gene expression analysis, could improve risk classification, and define patient prognosis.

We hypothesized that gene expression and DNA methylation of blasts obtained at diagnosis differ between MRD + and MRD- patients and by comparing these two conditions, candidate genes predictive of treatment response, relapse and death could be extracted. For this reason, we collected BM samples obtained at diagnosis, purified leukemic blasts and compared gene expression and DNA methylation profiles between MRD + and MRD- patients at day 15 and at the end of induction, looking for overexpressed and hypomethylated genes in MRD + patients. Subsequently, we evaluated if the selected genes could predict response to induction chemotherapy, relapse, or death. The search for new genomic biomarkers will improve risk classification and, in the future, patient survival.

Patient samples

Sixty-one patients with B-ALL who attended the Instituto Nacional de Cancerología, Hospital Militar Central and Hospital Universitario San Ignacio (Bogotá, Colombia) between 2017 and 2021 were included. The discovery cohort consisted of 19 BM samples taken at the time of the diagnosis in which RNA-seq/DNA methylation protocols were performed. Twenty-seven BM samples BM samples taken at the time of the diagnosis were included in the validation cohort by RT-qPCR. Fifteen BM from relapsed patients were include and only 1 of these patients had BM at the time of diagnosis, the others were patients who relapsed, were admitted to treatment centers and their BM was collected at that time.

Newly diagnosed patients were included in the study when they entered to the institutions for symptomatology associated with ALL and after verification of the inclusion criteria (not having received chemotherapy, not having another type of cancer, not having genetic diseases and being younger than 18 years old). The diagnosis was confirmed using flow cytometry (FCM) (19) and morphological analysis of BM. This study was conducted following the recommendations of the Colombian Regulation for Research in Humans (Resolution 8430 of 1993, Ministry of Health of Colombia) and in accordance with the Declaration of Helsinki and approved by each participating institution’s Institutional Review Boards. All methods for nucleic acid analysis were approved by the LSUHSC Translational Genomics Core’s Institutional Biosafety Committee protocol number 17370. Informed consent was signed by the parents of all participants. Each patient was treated according to the assigned risk and in accordance with the Berlin-Frankfurt-Munich protocol (BFM) (20). Patients with treatment abandonment or non-adherence to it were excluded.

According to the BFM protocol, response to induction therapy was evaluated by FCM detecting MRD at day 15, where patients with < 0.1% residual blasts in BM were classified as MRD- day15, and patients with > 0.1% residual blasts were considered MRD + day15. At day 33, patients with < 0.01% residual blasts in BM were MRD- day33, while patients with > 0.01% residual blasts were MRD + day33 (20). Therefore, we considered patients with MRD- day15 and MRD- day33 as true responders (MRD-/-) and patients with MRD + day15 and MRD + day33 as refractory patients (MRD+/+), this comparison was called a "true response”.

Blasts isolation and purification

BM samples (diagnosis and relapse) were collected by a hemato-oncologist and processed within 24 hours after sample collection. First, mononuclear cells were separated from BM by density-gradient centrifugation (Lymphoprep, Lonza). The blasts were separated using magnetic microbeads coated with anti-CD19 or anti-CD34 antibodies, followed by MACS column enrichment (Miltenyi, Bergisch Gladbach, Germany). The purity of sorted blasts was assessed with CD34-PERCPCy5.5, CD45-V500, CD19-PECy7, and CD10 APC antibodies. Data was acquired in a FACSCanto II flow cytometer (Becton/Dickinson Biosciences, San Jose, CA), using the FACSDiva software program. Infinicyt software (Cytognos SL, Salamanca, Spain) was used for data analysis (21).

DNA and RNA extraction

DNA and RNA were extracted from MACS-sorted blasts using the Allprep mini kit and robotic workstation QIAcube (Qiagen, Hilden, Germany). RNA quality was evaluated using the Agilent RNA 6000 Nano and Pico kits, and data were analyzed with Agilent 2100 Bioanalyzer. RNA concentration was calculated using the Qubit™ RNA High Sensitivity and Broad Range kits, while DNA concentration was calculated using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific).

Library preparation and RNA sequencing

Samples with RIN > 6 and purity by FCM with > 90% blasts were selected for RNA-seq. For RNA library preparation, 300 ng of total RNA was used. TruSeq Stranded mRNA RNA libraries were prepared following Illumina’s protocol. Resulting libraries were sequenced at 2 x 75 bp on a NextSeq550 sequencer system at the Stanley S. Scott Cancer Center’s Translational Genomics Core at LSUHSC-New Orleans. On average, more than 50 million reads were obtained per sample.

DNA methylation assay

Bisulfite conversion was performed in 500 ng of DNA for each sample following the recommendations of the EZ DNA Methylation-Startup Kit (Zymo Research, USA). Bisulfite-converted DNA was amplified and hybridized to the Infinium Methylation EPIC Kit chips and scanned on the Illumina’s iScan.

RT-qPCR

Total RNA from sorted blasts was treated with DNase I Amplification Grade (Invitrogen, USA) prior to reverse transcription. cDNA was synthesized using the SuperScript III First-Strand Synthesis SuperMix Kit (Invitrogen, USA), following the manufacturer’s procedures. TaqMan probes were used to quantify mRNA expression levels of candidate genes obtained by RNA-seq analysis (Assay IDs: DAPK1 Hs00234489_m1; NPDC1 Hs00209870_m1; CNKSR3 Hs00295109_m1; SLC18A2 Hs00996835_m1; CTHRC1 Hs00298917_m1; BOC Hs00264408_m1; SLC45A3 Hs00263832_m1; GAPDH Hs99999905_m1, ASCL2 Hs00270888_s1; MIR4435-2HG Hs03680374_m1). The reaction was amplified in a QuantStudio 12 K plex Real-Time PCR machine (Applied Biosystems). The 2^−ΔΔCT method was used to estimate the fold induction of each gene using GAPDH and Ct values to determine the fold change (FC) for each sample. A pool of samples was used as internal calibrator, as well as water as negative control. Assays were done in duplicate.

Statistical analysis

RNA-seq data quality control and filtering

The FASTQ files were uploaded to Partek Flow software to remove contaminants and align the reads to STAR 2.7.3 (reference genome hg38). Reads were quantified using RefSeq Transcrips 96. A quality analysis removed genes with zero or less than five reads. One sample with a low correlation (< 0.4) with respect to the others was removed.

Methylation DNA data quality control

Low-confidence probes (p-value > 0.05) and probes mapped to X and Y chromosomes were excluded. Normalization was done using NOOB (normalization for Illumina Infinium methylation arrays).

Transcriptomic and methylation data analysis

Normalization and differential expression analysis were performed using the Deseq2 library in RStudio. Differentially expressed genes (DEGs) were selected if they had a p-value < 0.05 and a FC > 2. Ggplot library was used to generate heatmaps, and GenomeStudio to calculate beta values for each hybridized probe. The Partek Genomic Suite was used for differential methylation analysis to identify and extract genes with differentially methylated CpGs (GDMCpGs). GDMCpGs were chosen if they had FC > 2 and FDR < 0.05. Enrichment analysis and functional gene annotation were performed using DAVID and Uniprot, respectively. Spearman correlation was used to determine correlation between overexpressed genes and hypomethylated probes; those with an inverse correlation less than − 0.50 and a p-value < 0.05 were selected.

Experimental design

Figure 1 describes the methodological design of the study. The gene and methylation profile of induction treatment were compared between MRD- vs. MRD + in each comparison group. Treatment response was the only variable used to define profiles in each comparison.

RT-qPCR analysis

Spearman correlation was used to determine any correlation between RNA-seq counts and FC and Ct values for RT-qPCR. Genes with p-value < 0.05 and r > 0.50 were selected. T-tests were used to compare FC between MRD- and MRD + in comparison groups and to compare FC MRD-/- and relapse samples. GraphPad software was used for statistical tests and graphic images.

Clinical data analysis

To compare clinical variables between patient cohorts, t-test and chi-square tests were used. The follow-up time for relapse and death was 2 years. Logistic regression analysis was performed to evaluate whether candidate genes could predict treatment response, refractoriness, or true response. Survival analyses were estimated according to gene expression using Kaplan-Meier curves. Cox regression was used to determine whether gene expression conferred a higher risk of death. The Youden index of normalized RNA-seq counts was used to define the cutoff threshold for overexpression for each gene. The Xena Browser (http://xena.ucsc.edu/) was used to observe the relationship between MIR4435-2HG, CNKSR3, and DAPK1 overexpression and survival (22, 23) using the following filters recurrent ALL samples of TARGET-Pan-Cancer cohort, ALL diagnosis, < 18 age, expression average of 7.82 for MIR4435-2HG, 10.58 for DAPK1, and 5.08 for CNKSR3, and 5-year overall survival.

Table 1 describes the clinicopathological characteristics of the patients included in the discovery cohort. It can be observed that there are no differences in clinical variables between MRD + and MRD- patients, except for risk. However, this was to be expected because MRD + patients are considered intermediate to high risk, whereas MRD- patients may be low to intermediate risk. Interestingly, more than sixty percent of patients had normal karyotype and the most frequent risk group was intermediate. In total, there were 2 relapses and 6 deaths. Deaths in the MRD- group were due to relapse and progression and another due to febrile neutropenia. In the MRD + group, there was also one death due to relapse and progression and 3 deaths during the induction phase (very aggressive disease).

Table 1

CLINICAL CHARACTERISTICS OF THE DISCOVERY COHORT
CLINICAL CHARACTERISTICS	MRD+ (n = 10)		MRD- (n = 9)
	n (%)	MEAN (RANGE)	n (%)	MEAN (RANGE)	p - value
AGE (years)		10.3 (3–17)		11.11 (3–15)	0.89
SEX
Female	4 (40)		3 (33.33)		0.76
Male	6 (60)		6 (66.67)		0.76
WBC (cel/µ)		58.36 (2.97–292)		53.56 (6.22–214.9)	0.89
RISK
Low	0 (0)		1 (11.1)		0.03
Intermediate	5 (50)		8 (88.9)
High	5 (50)		0 (0)
EXTRAMEDULLAR INFILTRATION
Yes	1 (10)		2 (22.2)		0.46
No	9 (90)		7 (77.8)		0.46
CORTICOID RESPONSE
Yes	9 (90)		9 (100)		0.32
No	1 (10)		0 (0)		0.32
CARIOTYPE/MOLECULAR ALTERATIONS
Normal	7 (70)		6 (66.6)		0.49
Hyperdiploid	1 (10)		0 (0)
t(1;19)	1 (10)		2 (22.2)
t(9;22)	1 (10)		0 (0)
t(3;14)	0 (0)		1 (11.2)
RELAPSE
Yes	1 (10)		1 (11.1)		0.93
No	9 (90)		8 (88.9)		0.93
DEATH
Yes	4 (40)		2 (22.2)		0.40
No	6 (60)		7 (77.8)		0.40
MRD + indicates minimal residual disease positive. MRD- indicates minimal residual disease negative.

To identify genes that could differentiate MRD + patients from MRD- patients, we separated with immunomagnetic column the leukemic blasts obtained at the time of diagnosis and obtained the gene expression and DNA methylation profiles of each sample. Subsequently, we collected clinical information from all patients to determine if they were MRD + or MRD-. Then, we compared the gene expression profiles and DNA methylation between MRD- and MRD + patients at day 15 of treatment, MRD- and MRD + patients at the end of induction and between MRD-/- vs MRD+/+ patients. Supervised hierarchical cluster analysis showed 159 DEGs at day 15 of induction treatment of which 127 genes were upregulated and 32 downregulated (Fig. 2a), 200 DEGs at the end of induction treatment of which 165 genes were upregulated and 35 downregulated (Fig. 2b) and 153 DEGs after comparison between MRD-/- and MRD+/+ patients of which 117 were upregulated and 36 downregulated (Fig. 2c). Finally, a Venn diagram was used to identify common DEGs between the three analyses, finding 50 of them (Fig. 2d). In MRD + patients, 46 of 50 common genes were up-regulated, and 4 of 50 were down-regulated (Additional file 1). Uniprot analysis showed that some genes were associated with different functions, including DNA binding (HOXB2, RFX8), transmembrane (SLC18A2, SLC45A3, BOC, ITGA3), lipidation (TNFRSF10C, PTGIR), among others. In addition, enrichment analysis showed that these genes are involved in processes like inflammatory response, transmembrane transport of sugars, cellular response to growth factor stimulus, etc.

To identify GDMCpGs, we used the same groups as in differential expression analysis. A total of 1834 GDMCpGs were obtained when comparing diagnosis BM of patients with MRD + and MRD- at day 15 (Fig. 3a); 2382 at day 33 (Fig. 3b); and 2077 between MRD+/+ and MRD-/- patients (Fig. 3c). We identified 687 common GDMCpGs across all comparison groups (Fig. 3d).

Then, to identify genes with both differential expression and methylation, we matched the previously obtained DEGs and GDMCpGs. We found 38 genes at day 15 of induction treatment, 52 genes at the end of induction, and 40 genes between patients with MRD+/+ and MRD-/-. Finally, a Venn diagram analysis identified 10 common genes (HOXB2, TMEM51, F2RL1, PDZRN3, BOC, TANC1, ASCL2, FBN2, MIR4435-2HG, and SCL18A2) (Fig. 3e). We found a significative inverse correlation between gene overexpression and hypomethylation in only four of them, which were present in all three comparison groups (BOC, MIR4435-2HG, SCL18A2, and ASCL2) (Table 2).

	Table 2. COMMON GENES BETWEEN THREE ANALYSIS GROUPS AND THEIR CORRESPONDS CpGDM
GENE/CpGs				MRD DAY 15		MRD DAY 33		TRUE RESPONSE
	FC	METHYLATION STATUS	RELATION TO CpGs ISLAND	p-value	Rho	p-value	Rho	p-value	Rho
HOXB2	5,1
cg22777724		Hypermethylation	S_Shore	0.45	0.2	0.08	0.47	0.08	0.49
cg09313705		Hypermethylation	S_Shore	0.32	0.26	0.18	0.37	0.45	0.22
cg22807449		Hypermethylation	S_Shore			0.18	0.53
TMEM51	5,4
cg24835051		Hypomethylation	Island			0.004	-0.73
cg15319451		Hypomethylation	Island			0.009	-0.67
cg13210325		Hypermethylation		0.09	0.43			0.54	0.18
cg07180646		Hypomethylation	S_Shore	0.08	-0.45
cg11618537		Hypomethylation						0.2	-0.37
F2RL1	3,5
cg12518146		Hypermethylation	Island	0.001	0.72	0.003	0.74	0.005	0.74
PDZRN3	5,4
cg04340156		Hypomethylation				0.27	-0.31
cg11461794		Hypomethylation				0.86	0.05	0.87	0.049
cg24353443		Hypomethylation	Island	0.66	-0.11			0.62	-0.14
cg05502701		Hypomethylation	Island	0.6	-0.14			0.45	-0.22
BOC	4,6
cg11205552		Hypomethylation				0.68	-0.11
cg22413784		Hypomethylation		0.05	-0.48	0.02	-0.58	0.01	-0.68
TANC1	3,5
cg21390755		Hypomethylation		0.11	-0.4	0.03	-0.57	0.12	-0.45
ASCL2	5,4
cg22346124		Hypomethylation	Island			0.13	-0.42
cg10290276		Hypomethylation	Island			0.03	-0.55
cg13930892		Hypomethylation	Island	0.004	-0.66	0.01	-0.65	0.02	-0.6
cg20912770		Hypomethylation	Island			0.05	-0.52
cg10526374		Hypomethylation	Island			0.05	-0.51
cg06263495		Hypomethylation	Island	0.01	-0.6	0.02	-0.59
cg26051413		Hypomethylation	Island					0.52	-0.19
cg11644479		Hypomethylation	Island					0.02	-0.62
cg13762320		Hypomethylation	Island					0.03	-0.58
cg19284039		Hypomethylation	Island					0.09	-0.48
FBN2	-6
cg23696863		Hypomethylation		0.05	0.49	0.09	0.46	0.12	0.45
MIR4435-2HG	4,4
cg24783876		Hypomethylation		0.02	-0.55	0.05	-0.51	0.06	-0.53
SLC18A2	8,7
cg03570973		Hypomethylation	N_Shore	0.001	-0.72	0.001	-0.77	0.0009	-0.8
cg19721867		Hypomethylation	Island			0.003	-0.72
cg10245915		Hypomethylation	Island			0.01	-0.65
cg01043119		Hypomethylation	Island			0.0005	-0.8
cg19617377		Hypomethylation	Island			0.003	-0.72
cg00512279		Hypomethylation	Island			0.001	-0.76	0.03	-0.58
cg08521987		Hypomethylation	Island			0.05	-0.51	0.007	-0.7
cg27186877		Hypomethylation						0.03	-0.59
Whitespaces indicate that not differentially methylated CpGs were identified in this comparison group. MRD, minimal residual disease. FC, fold change. Bold p-values indicate that CpGs are present in all three comparison groups and are statistically significant.

We selected six additional DEGs from the analyses between the MRD+/+ vs. MRD-/- comparison because it would allow the identification of specific genes of MRD+/+ patients, who have the worst prognosis; these genes were overexpressed and had GDMCpGs, and there was an inverse correlation between them. The resulting genes were DAPK1, NPDC1, CNKSR3, ITGA6, SCL45A3, and CTHRC1 (Additional file 2). Therefore, the 4 common genes between groups (BOC, MIR4435-2HG, SCL18A2, ASCL2) and the 6 genes derived from the MRD-/- vs MRD+/+ analysis (DAPK1, NPDC1, CNKSR3, ITGA6, SCL45A3, and CTHRC1) were selected for verification by RT-qPCR. All genes showed correlation between techniques except BOC and SLC18A2 (Fig. 4).

Due to the low incidence of the disease(24) and the small number of MRD + patients, samples from the MDR + patients in the discovery cohort, together with those from the validation cohort, were used for RT-qPCR analyses. Consistent with the RNA-seq results, MIR4435-2HG, CNKSR3, ITGA6, NPDC1, and DAPK1 were overexpressed in MRD + patients in all comparison groups and had differential FC values between conditions (Fig. 5). We used logistic regression to test if genes could predict response to induction chemotherapy. MIR4435-2HG was found to be the best predictor of whether a patient will be a MRD-/-, MRD+/+ (Figs. 6a-b), or MRD + at day 15 of induction (Figs. 6c-d). Regarding DAPK1, ASCL2, SCL45A3, NPDC1 and ITGA6, it was observed that they could also predict response at day 15 or whether the patient will be a true responder or refractory (Additional file 3 and 4); however, both MIR4435-2HG and the other genes do not predict whether the patient will respond at the end of induction (Additional file 5). To test whether MIR4435-2HG could also predict the risk of death, we performed logistic regression using our RNA-seq counts. We observed that MIR4435-2HG can predict death with good sensitivity and specificity (Fig. 6e-f). When analyzing the relationship between normalized RNA-seq counts of MIR4435-2HG and the probability of non-response to treatment at day 15 or being refractory, we found that patients with counts > 5.1 had a 66% probability of being refractory to treatment. This probability increased proportionally to gene expression. In addition, for patients with counts > 4.97, the probability of therapeutic failure at day 15 was > 60% and increased progressively as MIR4435-2HG overexpression increased. Similarly, the probability of death increased when counts were > 7.0 (Table 3).

Table 3

ANALYSIS OF PROBABILITY OF DEATH, REFRACTORINESS, FAILURE AT DAY 15 OF CHEMOTHERAPY INDUCTION ACCORDING TO MIR4435-2HG COUNTS
RNAseq COUNTS MIR4435-2HG	DEATH (%)	PROBABILITY OF REFRACTORY (%)	RNAseq COUNTS MIR4435-2HG	DEATH (%)	FAILURE RESPONSE DAY 15 (%)
4,42	32	1	4,36	15	25
4,45	33	2	4,52	17	33
4,55	33	3	4,54	17	34
4,56	33	3	4,64	18	40
4,74	35	10	4,64	18	40
4,89	36	24	4,78	20	48
4,91	37	27	4,81	20	50
5,15	39	66	4,81	20	50
5,15	39	66	4,85	20	53
5,55	43	97	4,96	22	59
6,05	49	100	4,97	22	60
6,31	51	100	5	22	62
7,2	61	100	5,14	24	69
9,44	80	100	5,21	25	73
			5,24	26	74
			5,35	28	79
			5,46	29	83
			5,59	32	87
			6,03	39	95
			6,06	40	96
			6,1	41	96
			6,23	43	97
			6,31	45	98
			7,18	62	100
			7,56	68	100
			8,09	77	100
			9,4	90	100

To test whether the identified genes could also be useful as prognostic biomarkers of relapse or death, first, we compared FC values between patients with MRD-/- and patients who relapsed using RT-qPCR analysis. Unfortunately, we did not have the diagnostic samples of the patients who relapsed, so we used the MRD-/- samples as the normal expression value because they have the best response to treatment. We found that DAPK1 and CNKSR3 had higher FC in relapse samples than in MRD-/- samples, while MIR4435-2HG showed a tendency (Fig. 7a). No difference was observed with ITGA6, NPDC1, ASCL2, CTHRC1 and SLC45A3 (Additional file 6).

Because in the previous analysis MIR4435-2HG only showed a tendency, we decided to analyze its expression in a different cohort using normalized RNA-seq counts obtained from the BM of 58 children with recurrent ALL from TARGET Pan-Cancer database. The 5-year survival analysis showed that MIR4435-2HG overexpression is a poor prognostic variable for patient survival (Fig. 7a). Interestingly, average counts in this cohort (7.82) correlate with our cutoff points, which would give a 100% probability of being refractory and an 80% probability of dying. Nevertheless, in this database, DAPK1 and CNKSR3 were not correlated with survival.

Four Cox regression models were performed to determine which variable might affect patient survival. The first model employed the clinical variables used currently to define the risk of death; in this model, no variable conferred risk of death. The second model used the same clinical variables and gene overexpression; in this model, no variable conferred risk either. However, in the third model, when using only MRD and the selected genes, MIR4435-2HG overexpression was the only variable that increased the risk of death 29 times. Similarly, in the fourth model, when evaluating the entire gene profile, MIR4435-2HG overexpression affected the risk of death (Table 3).

Table 3

MULTIPLE COX REGRESSION USING CURRENTLY CLINICAL VARIABLES AND GENE PROFILE
	MODEL 1. CURRENTLY USED CLINICAL VARIABLES		MODEL 2. CURRENTLY USED CLINICAL VARIABLES AND GENE PROFILE		MODEL 3. GENE PROFILE AND MRD		MODEL 4. GENE PROFILE
PARAMETER			p-value	OR (95%CI)	p-value	OR (95%CI)	p-value	OR (95%CI)
Age	0.76	NC	0.46	NC
WBC	0.62	NC	> 0.99	NC
Extramedullar infiltration	> 0.99	NC	> 0.99	NC
Corticoid response	0.15	NC	> 0.99	NC
MRD + day 15	0.36	NC	0.51	0.32 (0.002–11.91)	0.34	0.25 (0.006–3.80)
MRD + final induction	0.22	NC	0.36	3.71 (0.1786–121.0)	0.23	3.49 (0.357–37.17)
MIR4435-2HG			0.22	NC	0.01	29.42 (2.52-1 067)	0.005	16.47 (2.49–157.2)
DAPK1			> 0.99	NC	0.41	3.12 (0.16–96.95)	0.32	2.87 (0.26–24.31)
ITGA6			0.18	NC	0.11	0.03 (0.00002–1.59)	0.14	0.09 (0.002–2.27)
NPDC1			> 0.99	NC	0.10	13.22 (0.68–631)	0.09	9.40 (0.90–221.5)
CNKSR3			0.92	NC	0.87	1.17 (0.11–9.55)	0.97	0.97 (0.11–7.07)
ASCL2			> 0.99	NC	0.73	1.58 (0.08–29.20)	0.92	0.88 (0.05–11.05)
CTHRC1			> 0.99	NC	0.52	0.43 (0.02–5.49)	0.24	0.26 (0.01–2.56)
SCL45A3			> 0.99	NC	0.51	2.55 (0.08–45.41)	0.54	2.11 (0.11–24.78)
MRD + indicates minimal residual disease positive. NC, No calculable. WBC, white blood counts. Bold p-values indicate statistical significance (p < 0.05).

Strikingly, survival analysis showed that patients with MIR4435-2HG overexpression have worse survival; however, it is important to validate this result in a larger cohort of patients (Fig. 7b). However, because MRD is the most used variable to define risk of death, for survival analysis, we considered MRD at the end of induction, effectively showing that MRD- patients have better survival than MRD+ (Fig. 7c). Intriguingly, when we compared survival combining MRD + with MIR4435-2HG overexpression vs. MRD- with down-expression of MIR4435-2HG, we observed that these two variables separate the survival curves better, where patients with MRD + and MIR4435-2HG overexpression presented worse survival (Fig. 7d).

Finally, since more than half of our patients had normal karyotype, we wanted to test whether selected genes could improve risk classification in patients with normal karyotype. Surprisingly, we found that simultaneous overexpression of MIR4435-2HG, DAPK1, and ASLC2 was associated with poorer survival in these patients compared to those who did not overexpress them (Fig. 7e).

Since ALL are diseases with high heterogeneity at the genetic and epigenetic level, new biomarkers are needed to improve patient prognosis (25). The present study conducted an integrative analysis of genome-wide DNA methylation and gene expression by RNA-seq in a cohort of 19 pediatric patients with B-cell ALL to investigate whether differential DNA methylation genes and gene expression patterns could be proposed as potential predictive biomarkers that differentiate responder from non-responder patients and confer risk of relapse and death in pediatric patients with B-cell ALL.

Aberrant DNA methylation has been considered a hallmark in different types of cancer, including ALL (26, 27). As reported by Borssén et al, our study revealed a clear separation in both DNA methylation and gene expression profiles between MRD- and MRD + in all comparisons made; finding that overexpressed genes conferred a more aggressive phenotype (28). Previously, Figueroa et al, reported that aberrant DNA methylation in childhood ALL could be an important determinant of gene expression in disease-specific alterations (29). We observed correlation between hypomethylation of CpGs and overexpression of DAPK1, CNKSR3, MIR4435-HG2, CTHRC1, NPDC1, SLC45A3, ITGA6, ASCL2, BOC and SLC18A2 genes; demonstrating that changes in DNA methylation can influence gene expression.

Aberrant patterns of DNA methylation have been associated with clinical outcome in patients with ALL; however, additional research is required to assess the clinical utility of some of these findings (30–35). In this study, we selected MIR4435-2HG, DAPK1, ASCL2, SCL45A3, NPDC1 and ITGA6 as potential biomarkers of treatment response, whose overexpression predicts, with high sensitivity and specificity, treatment failure or refractoriness. Unfortunately, none of the genes predicted response at the end of induction, although this could be explained by the lack of a significant number of patients with MRD + for comparison. This analysis could be verified with a larger group of patients with MRD+. Nevertheless, we highlight MIR4435-2HG because its overexpression is the one that best predicts therapeutic failure.

Recently, selected genes had been described as possible diagnostic and prognostic biomarkers in different types of cancer and other non-neoplastic diseases (Table 4).

Table 4

LIST OF GENES CHOOSE AS POSSIBLE PREDICTIVE BIOMARKERS OF INDUCTION CHEMOTHERAPY RESPONSE
GENE NAME	FC	p-value	Rho	CpGs ASSOCIATED	RELATION TO CpGs ISLAND	PRESENT IN RESPONSE	CANCER ASSOCIATION	REF
DAPK1	3,9	0,000009	-0,91	cg11518830	S-shelf	MRD+/+	Gastric, pancreatic, head and neck, thyroid, brain, uterine, lung, esophageal cancers, CLL, AML and MDS	(36–44)
CNKSR3	5,7	0,0003	-0,85	cg00460149	Island	MRD+/+	Melanoma	(45)
SLC18A2	8,7	0,0009	-0,8	cg03570973	N-shore S-shore Island	MRD + day 15 MRD + day 33 MRD+/+	Prostate cancer and AML	(46,47)
CTHRC1	5,5	0,003	-0,76	cg01224234	S-shore	MRD+/+	Renal, head and neck, liver, stomach, lung, endometrial and colorectal cancers	(48,49)
NPDC1	4,4	0,003	-0,74	cg14190761	Island	MRD+/+	Gastric, neuroblastoma, pancreatic neuroendocrine tumors and AML	(50–53)
BOC	4,6	0,01	-0,68	cg22413784		MRD + day 15 MRD + day 33 MRD+/+	Gastric and pancreatic cancers	(54,55)
ASCL2	5,4	0,004	-0,66	cg13930892	Island	MRD + day 15 MRD + day 33 MRD+/+	Colorectal, gastric and lung cancer. Risk of ALL in pregnancy	(56–60)
SLC45A3	3,9	0,03 0,04	-0,59 − 0,57	cg01455178 cg04896348	N-shore S-shore	MRD+/+	Prostate cancer	(61)
ITGA6	3,8	0,02	-0,63	cg13586889	S-shore	MRD+/+	Multiple myeloma and AML	(62,63)
(64–68)	4,4	0,02	-0,55	cg24783876		MRD + day 15 MRD + day 33 MRD+/+	Digestive, reproductive, respiratory, nervous, and urinary tumors, AML and T-cell ALL	(64–68)

In particular, MIR4435-2HG, which is a long non-coding RNA, also named LncRNA-AWPPH, LINC00978, or MORRBID (65). Interestingly, MIR4435-2HG overexpression has previously been correlated with hypomethylation in gliomas (66). In patients with T-cell ALL, MIR4435-2HG was overexpressed compared to healthy patients, and has been shown to promote proliferation and inhibit apoptosis of ALL cell lines (69). Although the biological role of MIR4435-2HG is still under study, it is known to participate by deregulating different signaling pathways related to proliferation, invasion, migration, epithelial-mesenchymal transition, and apoptosis; specifically, participating in signaling pathways such as TGF-β, WNT-βcatenin, MDM2/p53, PI3K/AKT, Hippo, and MAPK/ERK (64, 66).

Unfortunately, no clinical variable was observed to increase the risk of death in our population; however, MIR4435-2HG overexpression increases risk, predicts death, and is associated with poorer survival. Similar results had been reported in acute myeloid leukemia (AML) by Zhigang Cai et al (70), who observed MIR4435-2HG overexpression and its association with poor survival in other cancer models, MIR4435-2HG overexpression has also been associated with worse progression-free survival and overall survival (64, 66, 67).

There are no studies that show the relationship between aberrant DNA methylation, gene overexpression, and treatment response, specifically at day 15 and the end of induction chemotherapy. However, some authors have found differences between the methylation profiles of patients who relapsed and those who did not (28), where less methylated CpG island methylator phenotype at diagnosis had an inferior overall survival compared to patients who had more methylated CpG island phenotype. Previously, Sandoval et al(71) reported hypomethylation in different genome regions, including Polycomb target genes, and its association with poor survival and relapse. Similarly, Hogan et al. reported epigenetic dysregulation in the acquisition of chemoresistance in relapse in genes CDKN2A, COL6A2, PTPRO, and CSMD1 (17). Here, we reported overexpression of MIR4435-2HG, DAPK1, and CNKRS3 in relapse samples, although the samples were not matched; in any case, it is still a surprising result because these genes are elevated in both non-responder and relapse diagnostic samples, indicating that they could be biomarkers of relapse. These results were also confirmed in the TARGET Pan-Cancer. Interestingly, in our study, 3 of 4 patients who died had MIR4435-2HG overexpression; these patients were MRD- at the end of induction and were intermediate risk but relapsed and died.

This could be a reason to implement a genetic panel, including MIR4435-2HG, especially when 25% of pediatric leukemia patients have no detectable genetic alterations and have a low mutation rate making risk classification difficult (72). In fact, 60% of our patients had no genetic alterations but overexpressed MIR4435-2HG, DAPK1 and ASCL2 correlating with worse survival, suggesting that expression of these genes could improve risk classification in these patients.

While MIR4435-2HG overexpression appears to be a poor prognostic factor, the association of DAPK1 expression with poor prognosis is controversial (73). In this study, we associated DAPK1 overexpression and hypomethylation with therapeutic failure, relapse, and poorer survival in patients with normal karyotype. However, in the Pan-Cancer analysis, patients with this overexpression had better survival. Therefore, there is no consensus on its prognostic role. DAPK1 has also been associated with resistance to imatinib in chronic myeloid leukemia (43), autophagia (74), alterations in the p53 signaling pathway in chronic lymphocytic leukemia (40) and methylated in myelodysplastic syndrome (38).

The limitations of this study are associated with the low number of newly diagnosed patients eligible for this investigation, as well as the limited number of MRD + patients. Another limitation is related to the follow-up period of the patients, in most cases the follow-up was 2 years, which does not allow us to generate a real conclusion of the observed results (relapse/death). For this reason, it is important to validate these findings in a larger cohort of patients with a longer follow-up.

Here, we propose a gene profile that can predict treatment response. Particularly, we demonstrate for the first time that MIR4435-2HG is overexpressed and hypomethylated in non-responder patients with B-cell ALL; it can predict treatment response and confers a higher risk of death to those patients who overexpress it. Its detection could be combined with MRD to improve risk classification in our patients, specifically those with normal karyotype and provide a new biomarker to evaluate post-treatment follow-up with the potential to be a useful predictive and prognostic biomarker in Hispanic children with B-cell ALL.

B-cell ALL B-cell acute lymphoblastic leukemia

ALL Acute lymphoblastic leukemias

MRD Minimal residual disease

BM Bone marrow

FCM Flow cytometry

BFM Berlin-Frankfurt-Munich

MRD+ Positive minimal residual disease

MRD- Negative minimal residual disease

MRD-/- MRD- day15 and MRD- day33

MRD+/+ MRD+ day15 and MRD+ day33

DEGs Differentially expressed genes

GDMCpGs Genes with differentially methylated CpGs

AML Acute myeloid leukemia

Ethics approval and consent to participate

This study was approved by the Comité de Ética en Investigaciones del Instituto Nacional de Cancerología (C-19010300404), the Comité de Investigaciones y ética del Hospital Universitario San Ignacio (2017/114) and the Comité de Ética en Investigación del Hospital Militar Central (2019-006). Informed consent was signed by the legal guardians of all participants.

Consent for publication

All authors have read and approved the manuscript and agree with submission to Biomarker Research.

Availability of data and materials: patient's clinical data may be requested to [email protected] under a justifiable reason. Methylation and gene expression data are available in the GEO repository under accession number GSE229056. Link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229056. Reviewers token: cdonecwstpcfvst.

Competing interests: The authors declare no competing interests.

Funding: This study was supported by the Instituto Nacional de Cancerología (C19010300-404).

Authors´ contributions

YTL: designed research, performed research, contributed vital analytical tools, collected data, analyzed, and interpreted data, performed statistical analysis, and wrote the manuscript. JZ: contributed vital new reagents and analytical tools, analyzed and interpreted data, performed statistical analysis, and wrote the manuscript. ALC: designed research, contributed vital new reagents and analytical tools, analyzed and interpreted data, and wrote the manuscript. PG: collected data, analyzed, and interpreted data, and wrote the manuscript. IDLR: collected data, analyzed, and interpreted data, and wrote the manuscript. NPG: collected data, analyzed, and interpreted data, and wrote the manuscript. LL: contributed vital analytical tools, analyzed, and interpreted data, performed statistical analysis, and wrote the manuscript. AI: collected data, contributed vital new reagents and analytical tools, and wrote the manuscript. NCR: designed research, analyzed and interpreted data, and wrote the manuscript. SQ: analyzed and interpreted data and wrote the manuscript.

Acknowledgments

We are mainly grateful to the patients who agreed to participate in the study, as well as to their tutors. We also thank the institutions that recruited the patients (Instituto Nacional de Cancerología, Hospital Militar Central and Hospital Universitario San Ignacio), especially Dr. Amaranto Suárez who provided support in the diffusion of the project within the Instituto Nacional de Cancerología, as well as Dr. David Garay, Dr. Eddie Pabón, Dr. Camila Prada, Dr. Jorge Buitrago, Andrea Naranjo, Giovanna Bedon, Bibiana Martinez, Paula Toro and Cindy Arevalo. Finally, special thanks to the Fundación Colombiana de Leucemia y Linfoma for the financial support to Yulieth Torres-Llanos in her PhD studies.

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No competing interests reported.

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MIR4435-2HG as a Novel Predictive Biomarker of Chemotherapy Response and Death in Pediatric B-cell All

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Patient samples

Blasts isolation and purification

DNA and RNA extraction

Library preparation and RNA sequencing

DNA methylation assay

RT-qPCR

Statistical analysis

RNA-seq data quality control and filtering

Methylation DNA data quality control

Transcriptomic and methylation data analysis

Experimental design

RT-qPCR analysis

Clinical data analysis

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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