Introduction
T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive form of ALL and represents 15–20% of pediatric ALL cases [
1]. Even with highly intensified therapy, 25% of T-ALL patients experience relapse and have lower post-relapse survival compared to the B-lineage ALL [
1]. The genetic heterogeneity of the disease makes T-ALL risk stratification difficult and hence all cases are treated upfront as high-risk with intensified therapy regimen. The high dose multi-agent chemotherapy is often associated with severe toxicities and long-term side effects. Thus, improving understanding of T-ALL biology through the identification and characterization of carcinogenic lesions is essential for better prognostic classification and treatment of the disease.
With the advent of next-generation sequencing techniques, many genetic abnormalities have been found in T-ALL over the last few decades. Aberrant expression of genes such as
LMO1, LMO2, TAL1, TLX1, TLX3, NKX2-1 and other transcription factors (TFs) have long been known [
2]. Whole-exome sequencing (WES), and RNA sequencing (RNA-seq) have extended the list of genetic abnormalities in T-ALL [
3,
4]. Besides aberrant expression that constitutes about 40–50% T-ALL [
5], RNA seq data has expanded the fusion gene list (30–40%) in T-ALL. Such fusion transcripts can either generate an over-expressing protein as in the case of
TAL1 as a result of
STIL::TAL1 fusion [
6,
7] or lead to over-expression of two truncated peptides such as
SET&
NUP214 in
SET:: NUP214 fusion [
8]. A number a novel fusion transcripts have been also identified in studies from different cohorts such as
ZBTB16::ABL1, RCBTB2::LPAR6, DLEU2::SPRYD7, TRAC::SOX8 etc. [
9,
10]. Further, in a holistic approach WES along with RNA seq has identified number of gene abnormalities in pathways regulating differentiation, proliferation, self-renewal, and survival of T-cell precursors. High mutation frequencies such as
NOTCH1, JAK-STAT, PI3K-AKT or
RAS-MAPK pathway genes have been noted in multiple studies although the frequency varies among cohorts and population being adult or pediatric [
11,
12]. Moreover, copy number variations (CNVs), especially high frequency of
CDKN2A/CDKN2B deletions have been consistently shown across multiple studies [
13‐
15].
Thus, the complex interplay of gene fusions, sequence aberrations and transcriptional expression profiles needs to be increasingly investigated in different cohorts to further refine current models of T-cell leukemia and to identify potential new biomarkers and therapeutic targets. In the current study, RNA-seq and WES analyses were performed in a prospective cohort of pediatric T-ALL cases. A number of rare gene fusions, mutations, aberrant transcripts, and CNVs were identified. Overall, our results point to the need for further large-scale genomic studies to improve patient stratification and optimize treatment strategies for pediatric T-ALL, especially in relation to our distinctly ethnic sub-continental population.
Discussion
Studying genetic alterations in T-ALL is the way forward in improving patients’ diagnosis and treatment. Being a genetically heterogeneous disease, multi-genomics approach needs to be applied across different cohorts to elucidate the genetics of T-ALL leukemogenesis. Different studies have used different approach to identify prognostic markers and targets of therapy in T-ALL such as whole transcriptomics studies, whole exome sequencing, targeted DNA sequencing etc. However, no data from Indian subcontinent has been reported till date from study comprising transcriptomic and genomic sequencing. In the current pilot study, we have applied whole RNA and DNA sequencing to unravel maximum genetic alterations both at genes as well transcripts level in Indian cohort of pediatric T-ALL patients. We found that each of the 25 cases of our T-ALL study population harboured at least one major genetic abnormality, including gene fusions, CNV, recurrent gene mutation and/or aberrant expression of genes that are key to leukemogenesis.
56% (14/25) of our T-ALL cases harboured fusion genes. As expected co-occurrence of fusion genes in the same case was not observed, suggesting their role as driver mutations. Further, fusions were noted to be coexisting with either point mutations or aberrant expression suggesting their possible cooperative effects.
STIL::TAL1 fusion being the most common (16%) in the current study, have been reported earlier by us and others in Indian cohort, however the frequency ranged from 18 to 27% [
24,
25]. Earlier reports were based mainly on multiplex ligation dependent probe amplification assay (MLPA) or RT-PCR. We are for the first time, studying the combined genetic heterogeneity in T-ALL cases from Indian cohort by RNA Seq.
We also noted a novel fusion of
ETV6::HDAC8 in our cohort. An interesting study by Fisher MH et al. has shown that cytoplasmic localization of ETV6 due to inherited mutation leads to over-expression of HDAC3-regulated interferon response genes that pre-disposes to malignancy [
26]. Fusions like
TCF7::SPI1 and LMO1::RIC3, are also rarely reported. One case carrying
TCF7::SPI1 fusion in a recent study cohort of 121 cases [
27]. Interestingly we also noted one case having
RCBTB2::LPAR6 fusion that has previously been reported in B-ALL and has been suggestive of partial loss of
RB1 gene [
9,
28].
In the current study we have demonstrated a number of genes with aberrant expression profile in transcription factors and related genes. The highest expression showing gene is
TLX3 which was noted in 30% of cases. Cryptic translocation of t(5;14)(q35;q32), have been shown to result over-expression of
TLX3 expression in pediatric T-ALL cases. Earlier studies have noted 20–25% cases of T-ALL with
TLX3 rearrangement/over-expression [
29,
30]. Further, this genetic aberration is also shown to be associated with
NOTCH1 mutation and/or
NUP214::ABL1 amplifications [
29]. Interestingly, all 7 cases with over expressed
TLX3 had
NOTCH1/FBWX7 mutation. Moreover, one cases that harboured
NUP214::ABL1 fusion in our subjects, had the highest expression of
TLX3. Over-expression of this case was noted with the fold change of > 7000 compared to controls. Further, over-expression of
TLX3 was noted to be significantly associated with
NOTCH1 mutations (
p = 0.04). However, when prognostic outcome was analyzed in such cases, the result was not significant.
Other noteworthy genes having aberrant expression in our cohort are
LMO1, RAG1, RAG2, NKX3-2, NKX3-1, MYB and
MYCN. Among these,
RAG2 and
MYB over expression showed a correlation with outcome parameters.
RAG2 over expresssion showed better EFS (
p = 0.01) and OS (
p = 0.01) in pediatric T-ALL patients. A recent study in cell lines and mouse model, has shown that
RAG1 and
RAG2 expression in both primary and transformed thymocytes is mediated by
NOTCH1 dimerization. Since many earlier studies suggest better outcome of patients with
NOTCH1 mutations [
31,
32], further investigation of
NOTCH1-RAG2 axis in T-ALL cells may provide indirect evidence of mechanism behind better prognosis. Over-expression of
MYB was also noted to associated with poor EFS (
p = 0.041) and RR (
p = 0.045). However, the case number over-expressing
MYB (
n = 4) was too small to draw any definite conclusion and further study in larger cohort is needed to validate the association.
Mutational analysis of T-ALL cases revealed most frequent mutation in
NOTCH1 gene as expected. Studies have shown the frequency of
NOTCH1 mutation in T-ALL from 50 to 70% [
33‐
35] while in Indian cohorts the frequency ranges from 40 to 50% [
36,
37]. The lower frequency observed in current population could be due to the smaller cohort size. In our cohort the 6 cases that had NOTCH1 mutation only one had relapse but no significant association was noted with any outcome parameters.
The frequency of
PHF6 mutation noted in our study population is 12% similar to other studies describing the range of 5–19% in pediatric patients [
37‐
39]. Somatic mutations and deletions of
PHF6 in pediatric T-ALL have been reported exclusively or predominantly in males [
13,
40]. In our cohort one out of 3 cases bearing
PHF6 mutation was female. Further, in the present study, 3 cases that had
PHF6 mutation did not have relapse or any other event in the median follow up of 22 months.
WT1 mutations were noted in 23% of current study cohort. Earlier studies have reported relatively lower frequency in
WT1 gene [
13]. In addition, we noted that all 3 cases having mutation in
WT1 gene coincided with mutations/deletions of
NOTCH1/FBXW7 or
PHF6 genes. Similar observation has been reported in an earlier study suggesting that loss of function in
WT1 gene may cooperate in disease pathogenicity of T-cell leukemia [
13].
We noted 5 cases with mutations occurring in
NRAS or
KRAS gene. Earlier studies have suggested
NRAS mutation as an independent predictor of a poor outcome in ALL but a few other studies have shown favorable prognosis of RAS mutation in T-ALL [
13]. In our cohorts 5 occurrence of
NRAS- or
KRAS mutation only one had relapse however, study on the larger cohort needs to be done before establishing any conclusion. Interestingly,
FLT3 mutation was noted in 2 cases in our study population. As a target of therapy such mutations may contribute in personalized treatment of patients.
CDKN2A/CDKN2B gene deletion has been most common genetic lesion in our population as reported previously by us and other groups in India [
24,
25]. Similarly, in the current study population too, we noted 88% of cases (15/17) to have
CDKN2A/ CDKN 2B deletion. Previous studies have shown that though it is the most common mutation in T-ALL with poor prognosis, it is suggested to be acquired during the course of leukaemia progression of T-ALL and is not a driver mutation of the cancer cells [
14]. However more studies are needed to utilize this gene as prognostic marker or target of therapy in future.
Although studies specially focused on pediatric T-ALL are scarce however, in the previous large studies based on pediatric T-ALL genomics and transcriptomic analysis, almost similar results have been reported. Masafumi et al., on analysis of 121 pediatric T-ALL patients reported similar results as our study where
NOTCH1 and
CDKN2A were the most frequently affected genes, they also reported
USP7 gene in which we did not find any mutation. They also documented a
SPI1 fusion and associated it with reduced overall survival, a finding we observed in our patient cohort too. However, we did not observe a statistically significant correlation, possibly attributable to our smaller sample size [
27]. In a separate interesting study that analyzed both pediatric and adult samples, common fusions identified in the pediatric population included
KMT2A, MLLT10, STIL-TAL1, and
LMO2 fusions. Additionally, commonly mutated genes in this population were
NOTCH1, KRAS, NRAS, and
CDKN2A. These findings closely align with our results [
41].
LEF1, WT1 and
BCL11B copy number abnormalities were reported from the TARGET study of 2471 pediatric cancer patients whereas in our group we found
CDKN2A, CDKN2B and
MTAP harboring most copy number variations [
42].
Thus, despite the major limitation of small cohort size of the study, we present relevant mutations and aberrant expression profile in pediatric T-ALL from Indian cohort. As ethnicity has been shown to be involved the variations in T-ALL genomics, our study is an addition of current genomics data sets available in pediatric T-ALL. Further, it will be interesting in future to study the non-coding mutations, such as microRNAs and lncRNAs to add to the cancer-related gene regulatory network changes underlying leukemogenesis of T-ALL.
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