ORIGINAL RESEARCH

published: 07 April 2022

doi: 10.3389/fmolb.2022.854098

Technical Validation and Clinical Utility

of an NGS Targeted Panel to Improve
Molecular Characterization of
Pediatric Acute Leukemia
Clara Vicente-Garcés 1,2†, Elena Esperanza-Cebollada 1,2†, Sara Montesdeoca 1,2,
Montserrat Torrebadell 1,2,3, Susana Rives 2,3,4, José Luis Dapena 2,4, Albert Català 2,3,4,
Nuria Conde 2,4, Mireia Camós 1,2,3 and Nerea Vega-García 1,2*
1
Hematology Laboratory, Hospital Sant Joan de Déu Barcelona, Esplugues de Llobregat, Barcelona, Spain, 2Leukemia and Other
Pediatric Hemopathies, Developmental Tumors Biology Group, Institut de Recerca Hospital Sant Joan de Déu, Esplugues de
Llobregat, Barcelona, Spain, 3Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud
Edited by: Carlos III, Madrid, Spain, 4Pediatric Hematology and Oncology Department, Hospital Sant Joan de Déu Barcelona, University of
Amit Prasad, Barcelona, Barcelona, Spain
Indian Institute of Technology Mandi,
India
Reviewed by:
Development of next-generation sequencing (NGS) has provided useful genetic
Frederik Damm, information to redefine diagnostic, prognostic, and therapeutic strategies for the
Charité Universitätsmedizin Berlin,
management of acute leukemia (AL). However, the application in the clinical setting is
Germany
Giovanni Cazzaniga, still challenging. Our aim was to validate the AmpliSeq™ for Illumina® Childhood Cancer
University of Milano Bicocca, Italy Panel, a pediatric pan-cancer targeted NGS panel that includes the most common genes
*Correspondence: associated with childhood cancer, and assess its utility in the daily routine of AL
Nerea Vega-García
nerea.vega@sjd.es
diagnostics. In terms of sequencing metrics, the assay reached all the expected
†
These authors have contributed
values. We obtained a mean read depth greater than 1000×. The panel demonstrated
equally to this work and share first a high sensitivity for DNA (98.5% for variants with 5% variant allele frequency (VAF)) and
authorship
RNA (94.4%), 100% of specificity and reproducibility for DNA and 89% of reproducibility for
Specialty section:
RNA. Regarding clinical utility, 49% of mutations and 97% of the fusions identified were
This article was submitted to demonstrated to have clinical impact. Forty-one percent of mutations refined diagnosis,
Molecular Diagnostics and while 49% of them were considered targetable. Regarding RNA, fusion genes were more
Therapeutics,
a section of the journal clinically impactful in terms of refining diagnostic (97%). Overall, the panel found clinically
Frontiers in Molecular Biosciences relevant results in the 43% of patients tested in this cohort. To sum up, we validated a
Received: 13 January 2022 reliable and reproducible method to refine pediatric AL diagnosis, prognosis, and
Accepted: 18 February 2022
Published: 07 April 2022
treatment, and demonstrated the feasibility of incorporating a targeted NGS panel into
Citation:
pediatric hematology practice.
Vicente-Garcés C,
Keywords: next generation sequencing (NGS), pediatric acute leukemia, clinical impact, molecular diagnostics,
Esperanza-Cebollada E,
precision medicine
Montesdeoca S, Torrebadell M,
Rives S, Dapena JL, Català A,
Conde N, Camós M and INTRODUCTION
Vega-García N (2022) Technical
Validation and Clinical Utility of an NGS
Acute leukemia (AL) is the most common pediatric neoplasm and the primary cause of death related
Targeted Panel to Improve Molecular
Characterization of Pediatric
to cancer in childhood. It is characterized by the clonal expansion of a myeloid immature progenitor
Acute Leukemia. (Acute Myeloid Leukemia, AML) or a lymphoid immature progenitor (Acute Lymphoblastic
Front. Mol. Biosci. 9:854098. Leukemia, ALL), with ALL being the most prevalent type of leukemia in children (Hunger and
doi: 10.3389/fmolb.2022.854098 Mullighan, 2015; Grimwade et al., 2016; Bolouri et al., 2017). The survival rates have improved

Frontiers in Molecular Biosciences | www.frontiersin.org 1 April 2022 | Volume 9 | Article 854098

Vicente-Garcés et al. NGS Validation for Pediatric Leukemia

significantly; however, an important proportion of patients still IDH1, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PDGFRA, PIK3CA,
relapse (Gatta et al., 2013). Personalized medicine for the PTEN, RET, and TP53.
treatment of AL provides a directed therapy for patients
based on the comprehensive analysis of different molecular
®
For the RNA analyses, we used SeraSeq Myeloid Fusion RNA
Mix (SeraCare, Mildford), which is a mixture of synthetic RNA
markers that can improve the diagnostic and prognostic fusions combined with RNA extracted from GM24385 human
algorithms. At the genetic level, pediatric cancers have reference line. We based the studies on ETV6::ABL1, TCF3::
distinctive features that make them different from adult PBX1, BCR::ABL1, RUNX1::RUNX1T1, and PML::RARA fusions.
cancers. Despite also finding gene fusions, copy number Negative controls were also needed. We used NA12878
variants (CNVs), insertions/deletions (InDels), and (Coriell Institute of Medical Research) as a DNA negative
epigenetic alterations, pediatric leukemia has a relatively control and IVS-0035 (Invivoscribe) as an RNA negative control.
low mutational burden, although generally clinically
relevant (Hiemenz et al., 2018). Many current clinical Patients
testing of these alterations are laborious, with multiple tests We selected 76 pediatric patients diagnosed with B-cell
performed separately for a single patient and alteration. In this precursors ALL (BCP-ALL) (n = 51), T-ALL (n = 11), and
setting, the development of next-generation sequencing (NGS) AML (n = 14) from different centers (Hospital Sant Joan de
techniques has made it possible to address the complexity of Déu, HSJD; Hospital Clínic de Barcelona, Hospital de la Santa
AL study (Mullighan, 2013; Izevbaye et al., 2020; Neumann Creu i Sant Pau, Hospital Jerez de la Frontera, Hospital
et al., 2754). NGS allows a parallel study of numerous genes Universitario Ntra. Sra. De Candelaria, Hospital Universitario
and patients with high sensitivity. Although a wide diversity of Miguel Servet, Hospital Universitario Cruces, and Hospital
commercial cancer gene panels is currently available for Clínico Universitario Virgen de la Arrixaca) from 2016 to
clinical practice, most of them are focused on adult 2020. Out of the 153 patients diagnosed in HSJD during this
patients. To supply this lack of pediatric NGS panels, some period, we selected those patients younger than 25 years old with
laboratories have developed NGS custom panels, but this is a available sample at diagnosis or relapse with high DNA and RNA
very laborious and time-consuming option. Therefore, many quality. We also applied a clinical selection criterion, using non-
laboratories choose to confirm that the commercially available consecutive samples and prioritizing those patients with non-
adult-focused NGS panels include also all the relevant genes defining genetic results using conventional diagnostic
for the pediatric approximation. Overall, despite the variety of methodologies that could benefit from NGS studies.
targeted panels to study different types of cancers, the Consequently, the genetic alterations frequencies from patients
availability of specific panels for pediatric AL is still limited. included in our study do not reflect the standard distribution of
™ ®
The AmpliSeq for Illumina Childhood Cancer Panel is a
pediatric pan-cancer NGS targeted panel specific for the study of
genetic abnormalities in pediatric acute leukemia.

the most common variants associated with childhood and young

adult cancer types. It analyses multiple variant types including
Molecular Characterization by
gene fusions, hotspot regions, single nucleotide variants (SNVs), Conventional Molecular Biology
InDels, and CNVs in more than one sample at the same time. Techniques
This study presents the validation of the AmpliSeq for ™ The mutational status of FLT3 (FLT3 internal tandem
®
Illumina Childhood Cancer Panel and its clinical utility in
daily routine in AL diagnostics. We assessed its sensitivity,
duplication, FLT3-ITD) and NPM1 was assessed by labeled-
PCR amplification as reported (Gale et al., 2008). FLT3
reproducibility, the limit of detection (LOD), and clinical tyrosine kinase domain mutations, cKIT, and GATA1
utility. Although this panel analyzes 203 genes involved in mutations were tested by Sanger sequencing (Kottaridis et al.,
different pediatric tumors, this validation is focused on those 2002; Rainis et al., 2003; Manara et al., 2021). The study of the
genes that are relevant for the diagnosis, prognosis, risk fusion genes CBFB::MYH11, RUNX1::RUNX1T1, PML::RARA,
stratification, or targeted therapy in pediatric acute leukemia. BCR::ABL1, ETV6::RUNX1, TCF3::PBX1, STIL::TAL1, KMT2A::
AFF1 (AF4), KMT2A::MLLT1 (ENL), and KMT2A::MLLT3 (AF9)
was done by quantitative RT-PCR (q-RT-PCR) using specific
MATERIALS AND METHODS primers and probes as specified in the Europe Against Cancer
Program guidelines (Gabert et al., 2003; Jansen et al., 2021).
Sample Selection
Commercial Controls Nucleic Acid Extraction and Quantification
Commercial controls were used to assess sensitivity and Nucleotide extraction was performed using different methods.
specificity and to establish the LOD. DNA was extracted with the Gentra Puregene kit (Qiagen,
As a positive control for the DNA analyses, we used SeraSeq
Tumor Mutation DNA Mix (v2 AF10 HC) (SeraCare, Mildford).
® Hilden, Germany), the QIAamp DNA Mini Kit, or the
QIAamp DNA 2.7 Micro Kit (Qiagen, Hilden, Germany).
This is a multiplex biosynthetic mixture of different clinically RNA was manually extracted using guanidine thiocyanate-
relevant DNA variants present at an average variant allele phenol-chloroform method (TriPure, Roche Diagnostics,
frequency (VAF) of 10%. The genes included are AKT1, APC, United States), or using column-based methods with Direct-
BRAF, CTNNB1, EGFR, ERBB2, FGFR3, FLT3, GNA11, GNAQ, zol RNA MiniPrep (Zymo Research, California, United States).

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Vicente-Garcés et al. NGS Validation for Pediatric Leukemia

The DNA and RNA purity were determined by Quawell according to the AMP/ASCO/CAP Standards and Guidelines
Q5000 UV-Vis spectrophotometer (Quawell Technology Inc., for Somatic Variant Interpretation and Reporting (Li et al.,
San Jose, CA), having all the samples an OD260/280 ratio >1.8. 2017). Turnaround time for this testing was approximately
Integrity was assessed by Labchip (PerkinElmer Inc., 3 weeks.
Courtaboeuf, France), and TapeStation (Agilent, Santa Clara,
CA). DNA and RNA concentration were determined by Variant Confirmation
fluorometric quantification using the Qubit 4.0 Fluorimeter Mutations found by NGS with VAF>15% were confirmed by
(ThermoFisher Scientific, Massachusetts, United States) with Sanger sequencing and fusions detected by the panel were
the dsDNA BR Assay Kit for DNA samples and the RNA BR confirmed by RT-qPCR, using ABL1 as a housekeeping gene.
Assay Kit for RNA samples. Different and specific primer sets were designed using
PRIMER3plus software (https://primer3plus.com/cgi-bin/dev/
primer3plus.cgi). For fusions, 500 ng of total RNA was
AmpliSeq™ for Illumina® Childhood Cancer retrotranscribed with the High Capacity Retrotranscription kit
Panel (Applied Biosystems).
Following a PCR-based protocol, the AmpliSeq for Illumina
Childhood Cancer Panel analyzes 203 genes per sample
™ ® To confirm the somatic or germline nature of the variants, we
used patient-matched bone marrow samples in complete
simultaneously. It includes 97 gene fusions, 82 DNA variants, morphological remission (CR) with negative measurable
44 full exon coverage, and 24 CNVs. residual disease (MRD), as assessed by 8-color flow cytometry.
In our study, we focused on fusion genes, SNVs, and InDels in
those genes related to AL. The full list of genes included in the Analytical Validation
panel is shown in Supplementary Table S1. The list of genes Run Metrics
involved in leukemia, and therefore assessed in the validation, are Acceptable sequencing metrics were established by measuring the
displayed in a different color in the same table. on-target and uniformity percentages and depth of coverage
across the samples sequenced. In accordance with the
Library Preparation and Sequencing manufacturer’s instructions, we expected to obtain >95%
Library preparation was performed using the AmpliSeq for ™ targets covered at a minimum of 500 × >90% of coverage
uniformity, and >80% of on-target aligned reads.
®
Illumina Childhood Cancer Panel kit (Illumina, San Diego, CA)
following the manufacturer’s instructions.
Briefly, a total of 100 ng of DNA was used to generate 3069 Accuracy
amplicons per sample, with an average size of 114 bp, covering Positive and negative commercial controls were used to define
coding regions of multiple genes. Simultaneously, 100 ng of RNA true positive (TP), false positive (FP), true negative (TN), and
per sample was used to study 1701 amplicons, with an average false negative (FN) values. All known variants from positive
size of 122 bp, targeting gene fusions. RNA was reverse controls were assigned as TP if they were detected and FN if
transcribed to cDNA using the Ampliseq™ cDNA Synthesis kit not. Negative control was a reference sample for different genes.
(Illumina Inc., San Diego, CA). Amplicon libraries, with specific This control enabled us to determine TN when no alteration was
barcodes for each sample, were generated by performing detected and FP when there was a variant called. Sensitivity and
consecutive PCRs. Quality controls (QC) were done after specificity were calculated from these values. To assess the
cleaning up the libraries. Finally, libraries were diluted to accuracy, we determined the values of sensitivity [=TP/(TP +
2 nM and then DNA libraries and RNA libraries were pooled FN)) and specificity (=TN/(TN + FP)]. The coefficient of
at a 5:1 ratio (DNA:RNA). The final pool was diluted to 17–20 variation (CV) was calculated to evaluate the reproducibility of
p.m. and sequenced on a MiSeq Sequencer with a MiSeq Reagent variant detection by two different persons. We considered
kit v3 (600 cycles) (Illumina, San Diego, CA). Four patients were acceptable a CV < 20%.
sequenced in each run, including paired DNA and RNA.
Limit of Detection
Data Analysis To determine the LOD, we sequenced different dilutions of the
Data obtained from the sequencer was analyzed using the DNA
amplicon app and RNA amplicon app (Illumina, Inc.) from
DNA and RNA commercial controls. The SeraSeq Tumor
Mutation DNA Mix was diluted with DNA from the NA12878
®
BaseSpace™ Sequence Hub. Data files were imported to negative control to obtain VAFs of 10, 5, 2.5, and 1.25%. For the
VariantStudio and then raw variants were filtered excluding ®
RNA study, SeraSeq Myeloid Fusion RNA Mix was diluted with
those with an ExAC population frequency of ≥1%, synonymous RNA from the IVS-0035 negative control to obtain different
variants, and those elsewhere apart from exonic regions. dilution ranges (10−2, 10−4, and 10−5).
Variants were visualized by using the Integrative Genomics
Viewer (IGV) to discard potential artifacts. The remaining Reproducibility
variants were manually curated and filtered using various Reproducibility was assessed by performing the libraries by two
databases including COSMIC (http://cancer.sanger.ac.uk/ different operators and sequencing them in two different runs.
cosmic), Varsome (https://varsome.com/), and ClinVar This enabled us to quantify the variation introduced by the
(http://www.ncbi.nlm.nih.gov/clinvar), and classified personnel.

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Vicente-Garcés et al. NGS Validation for Pediatric Leukemia

Determination of Clinical Impact Run Metrics

The clinical impact was determined based on the effect of the A total of 22 pools were sequenced in a MiSeq sequencer (20
biological findings of the panel on diagnosis, prognosis, and pools containing patients’ samples and 2 pools using different
potential treatment decision-making. Diagnosis classification dilutions of commercial samples). All the runs succeeded and
was based on the 2016 World Health Organization (WHO) completed the sequencing. The failure rate of sequencing for
integrated classification of patients. In addition, variants the RNA was 7.7% (7 samples out of 78), while in DNA it
known to contribute to the diagnosis of an AL new subtype was 0%.
were considered based on recent literature. The prognosis for Results obtained across the runs revealed an average read
de novo ALL patients was assessed following the risk per sample of 6.1x106 for DNA. The amplicon mean coverage
stratification used in the current national treatment was 2054× with a coverage uniformity of 97% (range,
protocol SEHOP-PETHEMA 2013. For AML patients, we 70.42–98.82%). A total of 17 amplicons were covered less
used the risk stratification of the NOPHO DBH AML 2012 than 100× and 87 less than 500× from a total of 3069
protocol. Those variants defining or contributing to patient amplicons. Overall, the percentage of amplicons covered >
risk stratification following new evidence were also considered. 500× was 97.2%. The on-target alignment was 96%. All these
Finally, clinical implications regarding treatment were parameters agreed with the manufacturer’s specifications.
evaluated according to the currently available evidence The most important genes involved in AL and included in
including the therapeutic protocol, as well as those variants the panel were covered above 500× with a depth range from
qualifying the patients for an FDA-approved therapy or 759.92× to 2529.8× and a mean coverage of 1868.92×
contributing to be enrolled in a clinical trial. Overall, results (Figure 1). Despite the high coverage of the genes, when we
were considered clinically useful if they affected and/or analyzed them in more detail, we found that 28 amplicons
contributed to one of these clinically determining factors, covering these genes had a read depth under 500×, and 3 of
corresponding to Tiers 1 and 2 (variants with strong or them < 100× (Supplementary Figures S1A,B). When
potential clinical significance) according to the AMP/ASCO/ reviewing these regions in detail, some of them cover
CAP guidelines (Richards et al., 2015). All cases were regions with low frequent point mutations such as IKZF1
individually reviewed by at least one hematologist and one exon 5, KMT2D exons 10, 19, 31, 39, PTEN exon 8, SH2B3
molecular expert. exon 2, and NOTCH1 exons 26 and 34. However, most of these
Somatic findings in some genes raised the suspicion of exons do not represent any known hotspot region for these
germline origin because the same variant can be both genes described in the literature.
somatic and germline. Variants were considered for further For the RNA, an average read per sample of 2.5 x 106 with a
testing if they met the following specific criteria; pathogenic/ mean coverage of 1,060x was obtained after sequencing all the
likely pathogenic variants with VAF > 30%, in genes known to samples. The 67.65% (range 50.04–98.34%) of the reads was
be associated with cancer or if the variant is a known founder aligned to the human genome.
mutation or if clinical features suggest a germline origin. In all
those cases VAF was carefully interpreted considering purity Accuracy
and ploidy of the sample, germline mosaicism, loss of The accuracy was estimated as the concordance between the
heterozygosity, CNV in
rearrangements, and read depth.
tumor cells, structural ™
variants detected by the AmpliSeq for Illumina Childhood
Cancer Panel and the variants reported by the commercial
®
Ethical Issues
®
samples’ distributors. The SeraSeq Tumor Mutation DNA
Mix and the NA12878 sample were used to define accuracy in
The study was conducted in accordance with the ethical DNA samples. Specificity and sensitivity were calculated at
standards and the Declaration of Helsinki. All samples were different VAF (10, 5, 2.5, and 1.25%).
stored in the HSJD Biobank and were used after informed In DNA samples with variants at 10% VAF, all the expected
consent was obtained either from the patients and/or from alterations were detected at the expected VAF (Figure 2), and
their legal guardians. no variants were identified in the known negative samples.
Thus, both sensitivity and specificity were 100%. When
analyzing samples with a 5% VAF, 1 FN was found but no
RESULTS FP were detected. These results showed 98.5% of sensitivity
and 100% of specificity. When we tried to detect variants at
Analytical Validation 2.5% VAF, the sensitivity decreased to 85.3% and the number
To perform the technical validation of the AmpliSeq for ™ of FN exceeded the TP, detecting 18 and 16, respectively. The
®
Illumina Childhood Cancer Panel, 80 samples corresponding
to 76 patients were sequenced (2 samples were sequenced twice
number of FP was zero, meaning that no variants were found
in negative controls and, thus, achieving a specificity of 100%.
and 2 were the negative and positive commercial controls). Finally, at a 1.25% VAF, we were able to detect only 13 positive
Additionally, the 4 commercial controls (2 positive controls cases out of 68 TP. In this case, sensitivity drastically decreased
from DNA and RNA as well as 2 negative controls from DNA to 19.12%, but the specificity remained 100%. Results are
and RNA) were diluted and were sequenced in 2 different runs. summarized in Table 1.

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Vicente-Garcés et al. NGS Validation for Pediatric Leukemia

FIGURE 1 | Mean read depth obtained for the different leukemia-related genes.

To assess the RNA accuracy, the SeraSeq Myeloid Fusion

RNA Mix and the IVS-0035 were used as positive and negative
® detected except one (PML::RARA), which was not detected by one
of the technicians, giving a sensitivity of 94.4% (Table 1). No gene
controls, respectively. For fusion detection in RNA samples, all fusions were detected in IVS-0035 RNA negative control. Thus,
®
gene rearrangements in SeraSeq Myeloid Fusion RNA Mix were specificity was 100%.

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Vicente-Garcés et al. NGS Validation for Pediatric Leukemia

FIGURE 2 | Comparison of the expected VAF and the mean VAF obtained for the different variants. For TP53 (c.267delC), the graphic only shows the value of the
reported VAF, as the commercial company did not specify the expected VAF for this variant.

For DNA, we found a reproducibility of 100%, as all the

TABLE 1 | Mean sensitivity across the studied variants obtained for DNA at
different VAF and for the undiluted RNA. alterations were called in both runs with a similar VAF (Figure 3;
Supplementary Table S2) and the CV was fewer than 20% in all
VAF Sensitivity (%) cases (range, 0–18% with a mean of 6%). When the samples were
DNA 10% 100 diluted, the results obtained by both operators were also
5% 98.5 reproducible in most of the cases.
2.5% 85.3 Regarding the RNA, to analyze the reproducibility we
1.25% 19.12
examined the same samples on two different runs performed
RNA Undiluted 94.4 by two different operators. Reproducibility was 89% for the
rearrangements tested, as Operator 1 was not able to detect
one of the fusion genes (Table 2).

Limit of Detection Clinical Performance

The LOD for DNA was assessed for SNVs and small InDels by Patients’ Characteristics
®
diluting SeraSeq Tumor Mutation DNA Mix into NA12878, After completing the technical validation study with commercial
obtaining different VAFs (10, 5, 2.5, and 1.25%), and then
determining the lowest VAF detectable. We were able to
controls, the clinical utility of the AmpliSeq for Illumina
Childhood Cancer Panel was evaluated using non-consecutive
™ ®
detect all the variants at 10% VAF, 98.5% of the variants at a clinical samples, priming the cohort in patients with unknown
VAF of 5%, 85.3% of the variants at 2.5% VAF, and 19% of the genetics and patients with not-stratifying genetics according to
variants at a 1.25% VAF. conventional methodologies.
To assess the LOD for RNA, the SeraSeq Myeloid Fusion ®
RNA Mix was diluted into IVS-0035, diluting the initial number
In total, 76 patients were included in the study. The median
age was 6.8 years (range, 0.22–25 years). Thirty-eight (50%) were
of copies to 10–2, 10–4, and 10–5 to determine the lowest number men and 38 (50%) were women. Overall, 62 corresponded to ALL
of fusion copies detectable. All the rearrangements were detected patients (51 BCP-ALL and 11 T-ALL) and 14 AML.
on the undiluted sample and the 88.9% (16 out of 18) on the 10–2
dilution. When the 10–4 and 10–5 dilutions were analyzed, no Mutation Distribution
rearrangements were detected (Table 2). After variant prioritization, 99 variants in 33 genes were
further considered as relevant in the 76 samples. The mean
Reproducibility number of mutations per sample was 1.3 (range, 1 to 7
The concordance of the different variants called between the runs mutations per sample). Thus, 68% of patients (n = 52)
performed by different laboratory technicians was used to decide showed at least one mutation. No mutations were detected
whether the performance of the libraries was reproducible. in 24 patients. All variants are detailed in Supplementary

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Frontiers in Molecular Biosciences | www.frontiersin.org

Vicente-Garcés et al.
TABLE 2 | Obtained reads, mean SD, and %CV for undiluted RNA and 10–2 dilution for each of the fusion genes analyzed. Libraries were performed by two operators (A and B).

Gene Id Hgvs Operator A Operator B Mean SD % CV

−2 −2 −2 −2
Undiluted 10 Undiluted 10 Undiluted 10 Undiluted 10 Undiluted 10−2
RNA reads dilution RNA reads dilution RNA reads dilution RNA reads dilution RNA reads dilution
reads reads reads reads reads

BCR::ABL1 BCR(NM_004327.3):r.1_3378 ABL 40,916 2014 40,376 1168 40,646 1,591 381.8 598.2 1 38
(NM_005157.3):r.83_5384
ETV6::ABL1 ETV6(NM_001987.4):r.1_737 40,128 672 15,024 678 27,576 675 17,751.2 4.2 64 1
(transcript 1) ABL1(NM_007313.2):r.576-5881
ETV6::ABL1 ETV6(NM_001987.4):r.1_1283 15,728 1052 20,890 676 18,309 864 3,650.1 265.9 20 31
(transcript 2) ABL1(NM_007313.2):r.576-5881
FIP1L1:: FIP1L1(NM_030917.3):r.1_1109 54,960 4104 44,730 1938 49,845 3021 7,233.7 1,531.6 15 51
>PDGFRA PDGFRA(NM_006206.5):r.2037_6590
7

MYST3:: MYST3(NM_006766.4):r.1_3803 20,532 1066 26,814 680 23,673 873 4,442.0 272.9 19 31
>CREBBP CREBBP(NM_004380.2):r.290_10197
PCM1::JAK2 PCM1(NM_006197.3):r.1_4365 17,866 974 26,152 736 22,009 855 5,859.1 168.3 27 20
JAK2(NM_004972.3):r.2008_5285
PML::RARA PML (NM_033238.2):r.1_1786_ Not detected Not 9,396 Not 9,396† — — — — —
ins134bp RARA (NM_000964.3): detected detected
r.657_3,301
RUNX1:: RUNX1 (NM_001754.4): r.1-803 11,028 856 12,968 462 11,998 659 1,371.8 278.6 11 42
>RUNX1T1 RUNX1T1 (NM_004349.3):r.419-7420
TCF3::PBX1 TCF3(NM_003200.3):r.1_1519 21,434 1842 23,426 868 22,430 1,355 1,408.6 688.7 6 51
PBX1(NM_002585.3):r.729_6918

SD: standard deviation; %CV: percent coefficient of variation.

†
This value is not a mean as it was detected by just one operator.
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NGS Validation for Pediatric Leukemia

Vicente-Garcés et al. NGS Validation for Pediatric Leukemia

FIGURE 3 | VAF obtained for the assessed genes performing the libraries by two different operators.

FIGURE 4 | Mutation and gene fusion distribution across the different patients analyzed.

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Vicente-Garcés et al. NGS Validation for Pediatric Leukemia

Table S3, and their distribution per gene and patient is shown uncertain clinical significance (TBLX1::TP63). Among the 29
in Figure 4. patients harboring clinically significant fusions, the genomic
Seventy-eight percent of variants (n = 77) were SNVs and 22% finding helped to refine the diagnosis or prognosis, and in 10
(n = 22) were InDels with a mean allele frequency of 41.73% cases (10/29, 34%) provided evidence for targeted therapy.
(range, 5–87.7%). Among the 203 genes of the DNA panel, 52 Overall, the clinical impact following international guidelines
genes were selected considering those previously reported as was 97% (29/30), meaning that most of the patients enrolled in
being involved in pediatric acute leukemia. Among these, 33 the NGS studies could benefit from the genomic findings derived
genes were mutated in our study: NRAS, KRAS, PTPN11, FLT3,
NOTCH1, ASXL1, RUNX1, TP53, ASXL2, MSH6, TPMT, PAX5,
™ ®
from the AmpliSeq for Illumina Childhood Cancer Panel
analysis. Of note, in 38% of patients (11/29), these molecular
PTEN, IKZF1, JAK1, NPM1, PRPS1, KMT2D, BRAF, NF1, markers would be missed by conventional molecular techniques
SMARCB1, MTOR, DDX3X, WT1, MET, ATRX, JAK3, (Figure 5).
FBXW7, NT5C2, IDH1, IDH2, APC, and STAT5B. Following a case review of each patient tested and the overall
A high rate of variants in genes involved in signal transduction clinical impact of comprehensive results for a given patient, 43%
and driver genes were observed. The most frequently identified of all patients derived a refined diagnostic, prognostic, and/or
variants were in NRAS (n = 14), KRAS (n = 10), PTPN11 (n = 7), therapeutic benefit from comprehensive genomic testing. In
FLT3 (n = 6), NOTCH1 (n = 6), ASXL1 (n = 3), RUNX1 (n = 4), BCP-ALL and AML, testing with the AmpliSeq ™
for
and TP53 (n = 3), all of them involving hotspot regions.
Mutations in genes involved in signaling pathways were the
®
Illumina Childhood Cancer Panel, most often impacted
patients by refining the diagnosis (64%). Moreover, genomic
most frequent and generally showed the lowest median VAF results were also particularly impactful for prognosis in 61% of
(5–30%) (NRAS, KRAS, PTPN11, and FLT3). these patients and therapeutic impact, evaluated in terms of
information for potential targeted therapy, was identified in
Fusion Distribution 48% of patients. In addition, 6% of patients, including both
We observed 15 different fusions in 30 of 76 patients. Fifteen BCP-ALL and AML, showed variants with suspected germline
patients harbored well-known frequent recurrent fusions variants (Figure 6).
currently employed to stratify patients in therapeutic Globally, considering DNA mutations and fusion genes, 83%
protocols, including ETV6::RUNX1, BCR::ABL1, KMT2A- of patients (63/76) showed at least one genetic alteration, and the
rearrangements, and RUNX1::RUNX1T1. The remaining NGS panel revealed additional molecular markers that could be
patients harbored already reported fusions that do not impact used for pediatric AL classification, prognosis, or treatment
our current treatment protocols (STIL::TAL1, RBM15::MKL1, selection. No genetic alteration was detected in 13 patients
PICALM::MLLT10) and novel fusions recently reported in the (cytogenetics of these patients is available in Supplementary
literature with potential or clinical impact in therapeutic Table S4). Finally, alterations in genes IKZF1, MSH6, RUNX1,
protocols [P2RY8::CRLF2, MEF2D::BCL9, MEF2D::CSF1R, TP53, and TPMT raised suspicion of germline predisposition in
ETV6::ABL1, TCF3::ZNF384, PAX5::NOL4L, RUNX1:: 13 patients, all of whom were referred to the cancer
CBFA2T3, RUNX1::USP42, and TBL1XR1::TP63 (Figure 4)]. predisposition program for genetic counseling and
All these fusions were considered clinically significant and confirmatory germline testing. However, we only confirmed a
were confirmed by RT-qPCR. The most frequent fusion was germline origin in genes MSH6, TP53, and TPMT in 5
ETV6::RUNX1 (n = 7), followed by P2RY8::CRLF2 (n = 5) and patients (6%).
STIL::TAL1 (n = 3).
Overall, 30 of 76 patients (39%) were found to carry fusions
with potential clinical impact. DISCUSSION
Clinical Impact of Genomic Findings The development of NGS in clinical laboratories has allowed a
As mentioned, compared to the conventional methodologies, the better characterization of leukemia leading to a better clinical
™ ®
AmpliSeq for Illumina Childhood Cancer Panel allows us to
analyze more genes or hotspots in a single assay.
management of the patients. However, the constant evolution of
this methodology, the wide range of panels and platforms, and the
A total of 99 variants were classified as pathogenic variants. lack of established universal standard quality criteria for NGS, its
Altogether, based on current AL clinical approved guidelines and application to a routine diagnostics laboratory needs to be
recently published studies, 49% (n = 48/99) of the variants were individually validated (Jennings et al., 2017).
clinically relevant. Furthermore, the variants were classified Here we describe the validation of a pediatric NGS cancer
according to their capacity to establish or refine the patient’s
prognosis and to identify associated targeted therapies. Based on
™ ®
panel, AmpliSeq for Illumina Childhood Cancer Panel, as well
as its clinical utility, in a cohort of 76 patients diagnosed with AL.
current protocols and recent literature, 70% of the variants could
be potentially used for pediatric acute leukemia risk stratification Analytical Validation
and 41% for treatment selection. The quality criteria established in the present study was based on
Fusions were present in 39% of patients (30/76), and all panel manufacturers’ expected yield, previous reports from
patients but one carried clinically significant fusions based on clinical laboratories that validated other NGS panels, and
currently published studies. Only one patient had a fusion of guidelines for NGS implementation in diagnostics (Richards

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Vicente-Garcés et al. NGS Validation for Pediatric Leukemia

FIGURE 5 | Clinical impact of panel sequencing regarding different genetic alterations.

FIGURE 6 | Clinical impact of panel sequencing by subtype of leukemia. (A) Overall clinical impact classified by the benefit apported. (B) Clinical impact and benefit
category depending on the different subtype of leukemia.

et al., 2015; De Leng et al., 2016; Jennings et al., 2017; Li et al., to those obtained in other panels or designs sequenced on
2017; Hirsch et al., 2018). In terms of sequencing metrics (mean Illumina platforms (Vega-Garcia et al., 2020). The assessment
read depth, uniformity, and so forth) the AmpliSeq for ™ of the mean read depth per amplicon showed good quality in
®
Illumina Childhood Cancer Panel performance in our
laboratory reached all the expected values and similar results
most of the analyzed fragments. The mean read depth obtained
overcame the expected, as we reached a mean greater than 1000×.

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Vicente-Garcés et al. NGS Validation for Pediatric Leukemia

These results technically validate the NGS assay and grant the leukemogenic genes and genes involved in signaling pathways,
identification of variant detection at high depth, which is which were also highly recurrent in our cohort (Holmfeldt et al.,
important when analyzing somatic tumor samples with 2013; Mullighan, 2013; Lindqvist et al., 2015; Paulsson et al., 2015;
variants at low VAF (Richards et al., 2015) and reaching a Tran and Hunger, 2020; Wang et al., 2020). The VAF analysis of
high level of sensitivity and specificity (see below). In our the variants per gene agreed with recent publications, suggesting
study, there were some regions from 5 different genes (IKZF1, that mutations in specific driver genes (NOTCH1, ASXL1, NPM1)
NOTCH1, KMT2D, PTEN, and SH2B3) where we did not obtain can be associated with clonal hematopoiesis. These mutations are
the expected read depth. Thus, when performing this NGS panel suggested to be acquired by preleukemic cells and can be stable
in clinical practice, it will be necessary to check those regions for years or, alternatively, can be acquired during leukemogenesis
using alternative techniques such as Sanger sequencing to ensure and be present in the founding clone (Young et al., 2016;
that there are no missed variants. Additionally, before Ferrando and López-Otín, 2017). On the other hand, genes in
implementing any NGS panel, it is necessary to confirm that it RAS-pathway tend to be subclonal (Jerchel et al., 2018). In this
includes all the genetic relevant variants for our disease of regard, the sensitivity of the NGS compared with conventional
interest. The AmpliSeq ™ ®
for Illumina Childhood Cancer
Panel covers most of the common variants observed in
techniques is especially important because patients harboring
these subclonal mutations could benefit from targeted therapy.
leukemia patients, as well as recently defined genetic subtypes. In the same line, some of the most recurrent fusion genes in
However, some rare or novel variants are lacking. One alternative pediatric leukemia were found in our cohort (ETV6::RUNX1,
to overcome this problem could be to customize the panel adding STIL::TAL1, KMT2A-rearrangements, RUNX1::RUNX1T1, BCR::
the absent genes, making the panel complete and adapted to our ABL1), as well as other novel rearrangements described, most of
disease. them associated with BCP-ALL B-other subtypes (MEF2D::BCL9,
™ ®
The AmpliSeq for Illumina Childhood Cancer Panel
demonstrated good sensitivity and specificity for both
MEF2D::CSF1R, ETV6::ABL1, TCF3::ZNF384, and PAX5::
NOL4L) or to rare and/or recent AML fusions reported in the
mutations and gene fusions. We achieved a sensitivity of 100% literature (RUNX1::CBFA2T3, RBM15::MKL1, RUNX1::USP42,
for variants with a 10% VAF and a sensitivity of 98.5% for and TBL1XR1::TP63) (Medinger and Passweg, 2017; Schwab
variants with 5% VAF. Moreover, for RNA, the sensitivity and Harrison, 2018; Inaba and Mullighan, 2020). Of note, the
achieved was 94.4%, and the specificity reached 100%. When performing for PML:RARA fusion was not reproducible, as only
filtering, the number of false positive variants can be reduced, one technician could detect this fusion. Regarding this, authors
increasing the specificity, by performing a visual inspection of reviewed the raw RNA data to look for the fusion and were able to
these variants using tools such as IGV. Although an LOD of 2.5% find it: it was probably missed by some parameters applied by
VAF for DNA was achieved in 85.3% of the cases, following Illumina RNA Amplicon algorithm analysis. This highlights the
international guidelines (Li et al., 2017) the cutoff threshold for need to validate the techniques in the laboratory before using
variant reporting was set at 5%, in which we were able to detect them to identify their weaknesses and, importantly, to integrate
95% of the samples. Thus, the implementation of NGS allowed us the molecular results in the clinical settings. In this particular
the detection of variants with low VAF and clonal heterogeneity, a case, if the initial clinical, morphological, and phenotypical
common feature in AL (Swaminathan et al., 2015; Ferrando and features raise the suspicion of acute promyelocytic leukemia,
López-Otín, 2017; Oshima et al., 2019). the emergency of the situation and the fast diagnosis needed
Finally, we demonstrated reproducibility of almost 100% with to discard the PML:RARA fusion would favor the use of the other
a CV fewer than 20% regarding DNA; for the RNA we obtained methodologies in the first place.
89% of reproducibility. Altogether, our data showed that, Despite the difficulty to incorporate all the relevant molecular
regardless of the technician processing the libraries, we were abnormalities described and remain updated, targeted panels are
able to detect the same variants in most of the cases. Thus, it was easy to integrate into clinical laboratories. In this regard, one of
not a hand-dependent finding, which is important when the main pitfalls of this panel is the absence of some important
implementing a new methodology in a routine clinical laboratory. genes such as NUTD15, MTHFR, and CEP72, as they are related
The number of variants per case was relatively low, reflecting to drug metabolism and response in new therapeutic protocols in
the low tumor mutational burden (TMB) observed in pediatric ALL (Maamari et al., 2020). Moreover, some interesting
tumors in contrast to adult tumors, except for those cases rearrangements such as MNX1::ETV6, DUX4-, ERG-
containing pathogenic variants in mismatched repair genes rearrangements, and IGH rearrangements (Inaba and
(Vogelstein et al., 2013; Gröbner et al., 2018). The general Mullighan, 2020; Quessada et al., 2021) are missed.
landscape of mutations observed in our cohort matches data
observed from recent large-scale pediatric leukemia studies. Clinical Impact
Somatic mutation and fusion distribution reflecting patterns of Regarding the clinical utility of the panel, overall, 49% of
co-occurrence and exclusivities and the suspected presence of mutations and 97% of the identified fusions were
germline predisposition were similar to other studies (Mullighan demonstrated to have a clinical impact by recent publications
et al., 2007; Inaba and Mullighan, 2020; Conneely and Stevens, and new proposals for pediatric treatment (Inaba and Mullighan,
2021; Klco and Mullighan, 2021; Quessada et al., 2021; Surrey 2020; JS and SE M, 2021). Regarding mutations, approximately
et al., 2021). In particular, the most significantly common 41% of them refined the diagnosis, while 49% were considered
mutated genes that these studies identified were driver targetable. On the contrary, fusion genes were more clinically

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Vicente-Garcés et al. NGS Validation for Pediatric Leukemia

impactful in terms of refining diagnosis (97%) than for applying

targeted therapies. In this sense, it is important to consider the
®
Illumina Childhood Cancer Panel is approximately 7 d for 4
patients in a MiSeq system. However, it could be reduced to 3–4 d
heterogenic complexity of overlapping findings within patients. analyzing only one sample in a MiniSeq system. The panel allows
Considering the number of patients who benefited from the us to adapt to a local setting and plan the application of a
targeted-NGS panel, our results showed that the results obtained combination of conventional and NGS approaches in a
™ ®
by the AmpliSeq for Illumina Childhood Cancer Panel were
clinically relevant in 43% of the patients tested in this cohort. This
stepwise manner. Now, there are few FDA-approved targeted
leukemia therapies available to pediatric patients, yet there are
rate of actionable alterations is similar to that detected in other novel leukemia subtypes in which targeted therapy (FDA-
published clinical sequencing studies in pediatric oncology, which approved and off-label) is accepted as best practice in the
showed between 30 and 60% rate of potentially targetable upfront or relapse setting. This includes the use of imatinib or
alterations and around 10% of germline mutations (Alonso dasatinib in ABL-class fusions patients or ruxolitinib for JAK2-
et al., 2019; Ishida et al., 2019; Oberg et al., 2021; Surrey et al., mutant patients (Roberts et al., 2014; Pui, 2020; Kantarjian et al.,
2021). Nevertheless, targeted panels contain a restricted number 2021). In our cohort, approximately 50% of patients would
of genes, usually the most mutated, which may make difficult the potentially have a targetable alteration supported by an FDA-
identification of rare or novel variants, as some of them could be approved drug or experimental drug with preclinical evidence
missed. To have a wider vision of the genetic landscape of the (approved with other indication or age). Furthermore, despite it
patients, it would be necessary to perform wider NGS strategies was out of the scope of this study, germline variant testing in
such as whole exome sequencing (WES) or whole genome TPMT and NUTD15 is essential given that certain
sequencing (WGS). However, these approaches require more polymorphisms in these genes lead to altered metabolism of
hands-on time, are more complex, and produce a huge the therapeutic agents and can inform about the requirement
amount of information, which translates into a longer analysis of adjusting the dosage of thioguanine and mercaptopurine
time and an unacceptable turnaround time to response in clinical (McLeod et al., 2000; Maxwell and Cole, 2017; Singh et al.,
practice. Therefore, choosing a targeted NGS panel adapted to
our disease is a good option, which can be combined with other
™ ®
2017). In this regard, the AmpliSeq for Illumina Childhood
Cancer Panel only allowed the identification of TPMT variants.
conventional methodologies that provide us complementary Somatic sequencing can also identify potential underlying
information for the diagnosis. germline variants and subsequent cancer predisposition
Panel testing in our cohort had an impact on refining syndromes, as some of the genes involved are the same for
diagnosis and prognosis in approximately 60% of the patients, leukemia patients (Obrochta and Godley, 2018; Bloom et al.,
especially in AML and BCP-ALL. However, if only those patients 2020; Klco and Mullighan, 2021). Both ALL and AML patients
with no genetic diagnosis by conventional methods were had a rate of around 10% of possible germline alterations
included, the expected clinical impact of the panel would be requiring additional confirmation and possible follow-up with
higher. The increasing knowledge on the genetic landscape of the cancer predisposition unit. In our cohort, the somatic
pediatric AL allows the proposal of new classifications based on mutations confirmed to be of germline origin, by testing them
molecular alterations that also can be used as markers for targeted in an MRD-negative remission sample were in TP53 and MSH6
therapy or follow-up of drug response based on MRD. In fact, genes, in line with the previous reports (Kraft and Godley, 2020;
new categories based on new rearrangements and mutational Klco and Mullighan, 2021). However, it is important to keep in
status have been added to the 2016 World Health Organization mind that this panel is not designed for this purpose, missing
classification (Arber et al., 2016; Wang and He, 2016). In this some important genes related to cancer predisposition (Desai
regard, the panel design can detect most of these novel mutations et al., 2017).
and rearrangements. Although some of the information gained In the aggregate, sequencing results provide extra valuable
from this panel could also be obtained through other means information clinically relevant for diagnostic, prognostic, and
(karyotyping, FISH, or qPCR), conventional approaches may be pharmacogenomics purposes, such as resistance alleles and
insufficient to stratify patients into recent proposals (Carbonell subclones, as well as information related to clonal evolution in
et al., 2019; Coccaro et al., 2019). Thus, the AmpliSeq for ™ the case of study-matched samples, where all of them do not meet
the definition of “actionability”.
®
Illumina Childhood Cancer Panel provides comprehensive
molecular analysis with increased efficiency avoiding the need
for staged molecular testing. Despite this, the need of combining
different diagnostic approaches including NGS must be carefully CONCLUSION
evaluated by each laboratory, especially to confirm/discard those
alterations that may modify the clinical management. Our study shows that targeted NGS-based assay is reproducible
Targeted NGS panels, such as AmpliSeqTM for Illumina
Childhood Cancer Panel, provide us with information of
® and precise; therefore, it is applicable in the pediatric AL
diagnostic workup after careful validation. Thus, the
hundreds of genes in a brief time, which is essential in clinical
practice. Depending on the local resources, the number of cases,
™ ®
AmpliSeq for Illumina Childhood Cancer Panel testing can
be efficiently incorporated into clinical care, providing useful
and the urgency to apply the results, it allows us to adapt the molecular information for diagnosis, prognosis, treatment, and
number of patients to be analyzed or the machine to run the follow-up. In our selected cohort of patients, the comprehensive
libraries. In general, the turnaround time for AmpliSeqTM for panel testing had a meaningful impact on clinical care, including

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Vicente-Garcés et al. NGS Validation for Pediatric Leukemia

diagnosis, prognosis, and treatment planning. Moreover, with the published version of the manuscript and NV-G is the
increase in precision medicine programs and the knowledge guarantor of this work and, as such, had full access to all data
acquired in integrating all the molecular data, the adoption of in the study and takes responsibility for the integrity of the data
a more inclusive definition of clinical impact would increase the and the accuracy of the data analysis.
benefits of incorporating NGS technologies in clinical practice.

FUNDING
DATA AVAILABILITY STATEMENT
Associations of parents and families of children with cancer from
The original contributions presented in the study are included in the Obra Social Hospital Sant Joan de Deu supported this work.
the article/supplementary material, further inquiries can be
directed to the corresponding author.
ACKNOWLEDGMENTS
ETHICS STATEMENT We thank the Hospital Clínic de Barcelona, Hospital de la
Santa Creu i Sant Pau, Hospital Jerez de la Frontera, Hospital
The studies involving human participants were reviewed and Universitario Ntra. Sra. De Candelaria, Hospital Universitario
approved by the Comitè d’Ètica d’Investigació amb medicaments Miguel Servet, Hospital Universitario Cruces, and Hospital
(CEIm) Sant Joan de Deu Fundació de Recerca. Written informed Clínico Universitario Virgen de la Arrixaca, for providing
consent to participate in this study was provided by the valuable patient samples. We are indebted to patients and
participants’ legal guardian/next of kin. families. We are grateful to Obra Social from Hospital Sant
Joan de Déu and many donors for their support. We also
wish to acknowledge “Biobanc de l’Hospital Infantil Sant Joan
AUTHOR CONTRIBUTIONS de Déu per a la Investigació,” integrated into the Spanish
Biobank Network of ISCIII for the sample and data
NV-G, EE-C, CV-G, and MC designed the study and MC and procurement.
NV-G supervised the project; SR, JD, AC, and NC recruited
patients. SM, MT, and MC performed the molecular diagnosis
and flow cytometry; CV-G and EE-C performed the libraries and SUPPLEMENTARY MATERIAL
the sequencing process; NV-G, EE-C, and CV-G performed the
bioinformatics analysis and analyzed the data; CV-G, EE-C, MC, The Supplementary Material for this article can be found online at:
and NV-G wrote the paper with the contribution of MT, SM, SR, https://www.frontiersin.org/articles/10.3389/fmolb.2022.854098/
JD, AC, and NC. All authors have read and agreed to the full#supplementary-material

Conneely, S. E., and Stevens, A. M. (2021). Acute Myeloid Leukemia in Children:

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