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\begin{document}

\title{Cancer Predisposition Syndromes in Children: Who, How and When Should
Genetic Studies Be Considered?}



\author[1]{Mónica Camacho}%
\author[1]{Marta Villa}%
\author[2]{Sara Álvarez de Andres}%
\author[3]{Bárbara Rivera}%
\author[1]{Paula Vázquez}%
\author[1]{Patricia Letón}%
\author[1]{Laura Martín}%
\author[1]{Marta Pilar Osuna-Marco}%
\author[1]{Blanca  López-Ibor }%
\affil[1]{Hospital Universitario HM Monteprincipe}%
\affil[2]{NIMGenetics Genomica y Medicina SL}%
\affil[3]{Institut d'Investigacio Biomedica de Bellvitge}%


\vspace{-1em}



  
  \date{March 13, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Early detection of cancer predisposition syndromes (CPS) is crucial to
determine optimal treatments and follow-up, and to provide appropriate
genetic counseling. This study outlines an approach in a pediatric
oncology unit, where 50 randomly selected patients underwent clinical
assessment, leading to 44 eligible for genetic testing. We identified 3
pathogenic or likely pathogenic variants in genes associated with CPS
and 6 Variants of Uncertain Significance (VUS) potentially associated
with cancer development. We emphasize the importance of a thorough and
accurate collection of family history and physical examination data and
the full coordination between pediatric oncologists and geneticists.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Unlike adult cancer, which is more commonly secondary to aging,
environmental exposures and lifestyle habits, childhood cancer is
remarkably different. No prominent causative factors have been
identified except for rare cases involving cancers attributed to viral
infections, radiation, oncological and immunosuppressive therapy. Recent
studies have identified a cancer predisposition syndrome (CPS) in 10\%
of children with cancer\textsuperscript{1-5}. The frequency of germline
variants is different between tumor types but appears to be more
significant in some malignancies such as central nervous system tumors
and paraganglioma\textsuperscript{6,7}.

In routine pediatric oncology practice, detecting these syndromes can be
challenging. There is a paucity of information regarding which patients
should be studied, when screening should be performed (e.g., at
diagnosis, at the end of the treatment), and which specific test should
be done, if whole exome sequencing (WES), trio-whole exome sequencing or
gene panels\textsuperscript{8}. Moreover, interpreting the genomic
results (based on established criteria\textsuperscript{9}) can be
overwhelming for clinicians.

Recent publications have reviewed the importance of early recognition of
these syndromes\textsuperscript{10-12} and clinical screening tools have
been proposed to help identify the patients who carry higher risk.
Particularly useful are the classic Jongmans
criteria\textsuperscript{13} and the recent McGill Interactive Pediatric
OncoGenetic Guidelines (MIPOGG)\textsuperscript{14-16}.

Genetic studies may not be universally accessible within all healthcare
systems. Consequently, it is crucial to prioritize the selection of
patients eligible for such studies. Heterogeneous strategies are
observed in various institutions, achieving similar
outcomes\textsuperscript{17-23.}

There has yet to be a consensus on the best time to carry out the
genetic study, whether at diagnosis, in the middle of, or at the end of
treatment. The most crucial advantage of performing the CPS study at
diagnosis is adjusting the patient's treatment to perform personalized
medicine. Avoiding radiotherapy in particularly sensitive patients
(e.g., Li Fraumenni patients\textsuperscript{24,25}), or choosing
targeted therapy specific to the patient's syndrome (e.g. immunotherapy
in patients with constitutional mismatch repair
deficiency\textsuperscript{26}), are some advantages of performing this
study early. Nevertheless, delaying the family cancer appointment could
provide an opportunity for more comprehensive information on
treatment-related toxicity and family medical history.

There is no consensus on which genetic study to perform, whether a WES
or a gene-targeted panel focusing on known genes associated with
CPS\textsuperscript{8}. Before the application of next-generation
sequencing (NGS), single-gene analysis of specific high-risk genes was
performed, a time-consuming and expensive approach. The widespread
availability and implementation of NGS significantly enhance the
efficiency of identifying individuals with CPS.

We present our experience in the recent years detecting patients with
CPS by multigene panel analysis. We highlight the importance of
dedicating a specific appointment with families to obtain a thorough and
accurate clinical history and to increase the efficiency of the process.

\textbf{Methods}

\textbf{Study design and participants}

Ethical approval was obtained through the regional scientific ethical
committee. Within the Pediatric Oncology department at HM Oncology
Center (CIOCC), HM Monteprincipe, all children with a malignancy, who
had finished their treatment were invited to participate in the study.
Patients who already had a diagnosis of a CPS as an early approach in
the acute phase or based on family history were excluded. A pediatric
oncologist trained in genetic counseling explained the procedure, as
well as its benefits and limitations. Informed consent was obtained from
the patient (if age-appropriate) and/or their parent/legal guardian.

Consented patients underwent a comprehensive cancer susceptibility
assessment with a pediatric oncologist, involving thorough physician
examinations, extensive three-generation family history, and collection
of tumor and somatic mutation data if available. Data were collected
based on established risk factors associated with CPS and were recorded
in a standardized form (Table 1). The malignancies included as
frequently associated with CPS are described in the supplementary file
(Table 1 of supplementary material).\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\textbf{Family History} & \textbf{Tumor} & \textbf{Patient
Characteristics}\tabularnewline
Two or more neoplasia in family members before the age of 18 (including
index case). Parents or siblings with cancer before age 45 Two or more
1st or 2nd-degree relatives degree relatives in the same family branch
diagnosed with cancer before the age of 45. Consanguinity of the
patient's parents & 1. Diagnosis of a type of tumor frequently
associated with cancer predisposition syndromes 2. Finding a previous
mutation in the tumor analysis suggesting a germline predisposition
syndrome 3. Patient with 2 or more tumors (bilateral, multifocal,
metachronous, or secondary tumor) & 1. Phenotype compatible with a
cancer predisposition syndrome 2.Patients with high toxicity to cancer
treatment (toxicity not expected with the type, degree, or duration of
the treatment received)\tabularnewline
\bottomrule
\end{longtable}

\textbf{TABLE 1.} Data collected in the familial cancer appointment.

If the patient met at least one criterion (listed on Table 1), blood
samples were collected from the child and both parents. A targeted exome
sequencing based on a panel of 110 genes frequently associated with
childhood cancer was performed on the patient. The genes studied are
shown in the supplementary file (Table 2 of supplementary material).

In each patient, we additionally gathered the same data (physical exam
and family history) directly from the patient's medical record before
this scheduled visit (''pre'' data). Our objective was to assess whether
the structured approach, in a dedicated medical appointment, and
consistent data collection by the same oncologist or a trained
associate, influenced the study's selection criteria outcomes.

Retrospectively, we have validated our selection criteria by comparing
them with the results we would have obtained with one of today's most
widely used tools, MIPOGG\textsuperscript{14-16}.

\textbf{Whole exome sequencing}

Genomic DNA from the proband's blood was isolated with an automatic
system (MagnaPure, Roche). A library was prepared using Comprehensive
Exome Panel technology (Twist Bioscience), sequenced on the NovaSeq 6000
SystemTM (Illumina) and designed with the Comprehensive Exome Panel
technology (Twist Bioscience) to capture over 20,000 genes, achieving
more than 99\% coverage of genes included in the RefSeq, CCDS, and
GENCODE databases, encompassing over 85\% of gene-mediated
disease-related alterations and flanking splicing regions (5-20 bp). The
library size is 41.2 MB. These processes were carried out at Nimgenetics
laboratory.

Data alignment and calling with the reference genome (GRCh38/hg38) in
the target regions (Twist\_Exome\_RefSeq\_targets\_hg38.bed) was
performed with DRAGEN\textsuperscript{TM} software version
07.021.572.3.6.3. Variant annotation was carried out using custom
software developed using NIMGenetics database and free publicly
available sources. The analysis focused on identifying variants in
exonic regions or splicing regions (at least 5 bp), including missense
or nonsense mutations, synonymous mutations, small insertions or
deletions (indels) with an allele frequency (VAF) greater than 30\% of
the reads. Identified variants were cross-referenced with specific
databases like ClinVar for known phenotype associations and population
frequency databases (dbSNP, gnomAD, 1000 Genome Project, or NHLBI-ESP
6500 exomes) to annotate variants commonly found in the general
population (at least 1\%). Pathogenicity of variants was estimated using
CADD and a combination of prediction systems from the dbNSFP database
(SIFT, PolyPhen2, MutationTaster, MutationAssessor, LRT, FATHMM, and
MetaSVM) for missense mutations.

The impact of mutations in splicing regions on mRNA processing was
evaluated using SpliceSiteFinder and MaxEntScan prediction systems.
Nucleotide position conservation was assessed using UCSC score ranges
from the PhyloP tool. The association of identified variants with OMIM
syndromes was evaluated. The nomenclature and classification of variants
follow the guidelines of the Human Genome Variation Society and the
American College of Medical Genetics and Genomics. Geneticists and
clinicians jointly interpreted the data in a multidisciplinary
assessment process before issuing the final report.

\textbf{Results}

\textbf{Description of the cohort}

A random sample of 51 patients who had already finished treatment in our
unir were approached for participation in this study. Acceptance was
98\% with just one family refusing to participate. The 50 initial
patients (ages ranging from 2 months to 22 years) were recruited from
October 2019 to April 2022.

A detailed clinical description of the cohort is summarised in Table 2.
The malignancies or hematologic problems are detailed in Table 3.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\textbf{Total number of patients} & \textbf{N} &
\textbf{\%}\tabularnewline
& 50 & 100\tabularnewline
\textbf{Sex} & &\tabularnewline
Female & 20 & 40\tabularnewline
Male & 30 & 60\tabularnewline
\textbf{Age at diagnosis} & Median age ( 7,09 years) & Median age ( 7,09
years)\tabularnewline
0-5 years & 24 & 48\tabularnewline
6-10 yr & 12 & 24\tabularnewline
11-15 yr & 9 & 18\tabularnewline
16-22 yr & 5 & 10\tabularnewline
\textbf{Diagnosis} & &\tabularnewline
\textbf{Hematologic tumor} & \textbf{9} & \textbf{18}\tabularnewline
\textbf{Solid tumor} & \textbf{41} & \textbf{82}\tabularnewline
\textbf{Treatment} & &\tabularnewline
Systemic treatment & 21 & 42\tabularnewline
Combined (Systemic+radiotherapy) & 21 & 42\tabularnewline
Radiotherapy & 2 & 4\tabularnewline
None & 6 & 12\tabularnewline
\bottomrule
\end{longtable}

\textbf{TABLE 2.} Epidemiology. Systemic treatment includes
chemotherapy, immunotherapy, immunosuppressive treatment, and
hematopoietic stem cell transplantation. Non-systemic or radiotherapy
refers to patients with surgical approaches exclusively.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\textbf{Diagnosis} & \textbf{50} & \textbf{100\%}\tabularnewline
\textbf{Hematologic tumor} & \textbf{9} & \textbf{18}\tabularnewline
Acute lymphoblastic leukemia & \textbf{5} &\tabularnewline
Non-Hodgkin Lymphoma & \textbf{2} &\tabularnewline
Myelodysplastic syndrome/aplastic anemia & \textbf{2} &\tabularnewline
\textbf{CNS tumor} & \textbf{10} & \textbf{20}\tabularnewline
Medulloblastoma & 2 &\tabularnewline
Ependymoma & 3 &\tabularnewline
Glioma & 3 &\tabularnewline
Schwannoma & 1 &\tabularnewline
Glial neuroepithelial & 1 &\tabularnewline
\textbf{Sarcoma/Rhabdomyosarcoma} & \textbf{17} &
\textbf{34}\tabularnewline
Osteosarcoma & 3 &\tabularnewline
Rhabdomyosarcoma & 8 &\tabularnewline
Ewing sarcoma & 4 &\tabularnewline
PNET & 2 &\tabularnewline
\textbf{Non-CNS embryonal tumor} & \textbf{10} &
\textbf{20}\tabularnewline
Neuroblastoma & 7 &\tabularnewline
Nephroblastoma & 2 &\tabularnewline
Hepatoblastoma & 1 &\tabularnewline
\textbf{Other} & \textbf{4} & \textbf{8}\tabularnewline
Lung neuroendocrine tumor & 1 &\tabularnewline
Non-Langerhans cell histiocytosis & 1 &\tabularnewline
Leydig cell tumor & 1 &\tabularnewline
Melanoma & 1 &\tabularnewline
\bottomrule
\end{longtable}

\textbf{TABLE 3} . Description of the tumors presented by our patients.
Abbreviations: CNS: Central Nervous System. PNET: Primary peripheral
neuroectodermal tumor.

\textbf{Patient selection}

We scheduled specific appointments with families to collect
comprehensive information about the family's cancer history, any
available molecular tumor data, and physical examination. If they met
any criteria outlined in Table 1, we conducted genetic testing on the
patient and their parents. Out of 50 patients, 44 (88\%) met at least
one criterion and underwent genetic testing, while the remaining 6
patients (12\%) were excluded from the study. Results and workflow are
shown in Fig.1.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} Workflow and results of selected patients.

Of the patients, 47,7\% met only one criterion, 40,9\% met two criteria,
9,1\% met three criteria, and 2,3\% met four criteria. 16 patients were
selected by family history (32\%), 31 patients (50\%) were selected by
tumor type (24 by tumor diagnosis and 7 by the presence of a somatic
variant detected in the tumor). 14 patients (28\%) met the criteria for
a compatible phenotype, and 11 patients (22\%) due to experiencing high
toxicity during treatment.

The patients selected by mutations present in the tumor were:

\begin{itemize}
\tightlist
\item
  High-grade neuroepithelial tumor with variants in BAP1 and BCOR in
  tumor.
\item
  Two patients with neuroblastoma and ALK somatic variant identified in
  the tumor.
\item
  Patient with osteosarcoma a TP53 somatic variant identified in tumor
\item
  Patient with medulloblastoma and a TP53 somatic variant identified in
  tumor
\item
  Patient with parameningeal rhabdomyosarcoma that presents in the tumor
  an NF1 rearrangement and a BCOR loss.
\item
  Patient with schwannoma and deletion of FANCL in tumor.
\end{itemize}

''Pre-data'' and ''post-data'' information were compared. Interestingly
24\% of patients did not meet any criteria on the ``pre-data''
evaluation (at diagnosis), but did meet at least one criterion on the
specific appointment for familial cancer (post-data) (Figure2).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2. Inclusion criteria pre-data and post-data. a.} Number
of patients that change their criteria in the pre-data evaluation
(diagnosis) and in the post-data (specific appointment)\textbf{.
b.}Differences between the criteria met at diagnosis (pre-data) and the
specific appointment (post-data).

In comparing the selection criteria with the MIPOGG tool, patients who
did not meet any criteria in our study similarly did not meet criteria
with MIPOGG. However, there are 9 patients whom the MIPOGG tool would
not refer for genetic study, but who were investigated according to our
criteria. Overall, the concordance rate was 82\%.

\textbf{Genetic Variants Identified}

Out of the 50 patients, in 3 (6\%), pathogenic or likely pathogenic
genetic variants that could explain their phenotype were found. In 6
patients (12\%) we identified a VUS, which after reviewing the published
data on the variant/gene and the patient's clinical history, we found
that the variant could be related to the patient's phenotype (Fig. 3,
Table 4). Overall, variants potentially associated with cancer
development were identified in 18\% of the studied patients.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3} . Overview of pathogenic/likely pathogenic variants
and VUS associated with phenotype. We identified 3 pathogenic or likely
pathogenic variants in genes associated with cancer predisposition
syndrome as well as 6 VUS potentially associated with the development of
cancer in these patients.\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
\begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\textbf{Diagnosis (age)}\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\textbf{Gene/} \textbf{transcript}\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\textbf{Chromosomal position in bp (hg19)}\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\textbf{Chromosomal position in bp (hg19)}\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\textbf{Nucleotide /aminoacid change}\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\textbf{Category}\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\textbf{Zygosity}\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\textbf{Origin}\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\textbf{Clinical signs/Comments}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Neuroblastoma (1 month)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{NM\_000059.3}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{chr13} \emph{g.32914515}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{c.6024dup} \emph{p.Gln2009Alafs*9} \emph{A/AG}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{c.6024dup} \emph{p.Gln2009Alafs*9} \emph{A/AG}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{Pathogenic}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{Heterozygous}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{Mother}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{Family history of breast cancer}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{Wilms Tumor} \emph{(22 y)}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
NM\_004484.3\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
chrX g.132838281\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
c.1033-4225G\textgreater{}A T/T\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
c.1033-4225G\textgreater{}A T/T\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Likely Pathogenic\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Heterozygous\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Mother\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Schwannoma (6y)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
FANCL\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
chr2 : 58386860-58390670\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
9-14 Exon deletion\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
9-14 Exon deletion\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Pathogenic\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Heterozygous\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Presented as well in the tumor and detected by MLPA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Glial neuroepitelial Brain Tumor (2 y)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{NM\_004656.3}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{chr3} \emph{g.52438516\_52438518}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{c.1201\_1203delinsGAG} \emph{p.Tyr401Glu}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{c.1201\_1203delinsGAG} \emph{p.Tyr401Glu}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{VUS}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{Heterozygous}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{Father}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{Presented as well in the tumor. BCOR mutation in the tumor}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{Neuroblastoma} \emph{(18 months)}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
NM\_006218.3\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
chr3 g.178916677\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
c.64G\textgreater{}A p.Val22Ile\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
c.64G\textgreater{}A p.Val22Ile\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
VUS\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Heterozygous\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Father\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
ALK mutation in tumor\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Neuroblastoma (15 months)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{NM\_004304.4}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{chr2} \emph{g.29498276}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{c.1904A\textgreater{}G} \emph{p.Tyr635Cys}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{c.1904A\textgreater{}G} \emph{p.Tyr635Cys}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{VUS}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{Heterozygous}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{Mother}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{Rhabdomyosarcoma} \emph{(6 y)}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
NM\_000076.2\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
chr11 g.2906145\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
c.575C\textgreater{}G p.Pro192Arg\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
c.575C\textgreater{}G p.Pro192Arg\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
VUS\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Heterozygous\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
De novo\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Neuroblastoma (7 months)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{NM\_002524.5}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{chr1} \emph{g.115252309}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{c.331A\textgreater{}G} \emph{p.Met111Val}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{c.331A\textgreater{}G} \emph{p.Met111Val}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{VUS}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{Heterozygous}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{Father}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{Wilms Tumor} \emph{(13 months)}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
REST \emph{NM\_001193508.1}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{chr4} \emph{g.57796160}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{c.1136A\textgreater{}G} \emph{p.His379Arg}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{c.1136A\textgreater{}G} \emph{p.His379Arg}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{VUS}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{Heterozygous}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{Father}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{TABLE 4} . Pathogenic or likely pathogenic variants and VUS
associated with phenotype.

Among the 9 patients in whom we identified pathogenic/likely pathogenic
or VUS likely associated with the phenotype, 8 (88.8\%) met the tumor
type criteria. This finding aligns with previous studies highlighting
the significance of tumor type in the suspicion of a
CPS\textsuperscript{6,7}.

Among the seven patients studied for tumor mutation, in two patients
(28.57\%) such mutation was found to be germline. This was observed in
the patient with somatic variant in BAP1 and BCOR in tumor (both
germline and somatic mutations were identified in BAP1), and the patient
with exon deletion of FANCL in tumor (the same deletion was found in
germline by MLPA).

\textbf{Discussion}

This study outlines an approach to investigating familial cancer within
a pediatric oncology unit from October 2019 to April 2022. Before
initiating this project, only patients with a substantial family history
or highly distinctive phenotypes were subject to investigation. The
primary change implemented in the study was the establishment of a
dedicated appointment for familial cancer assessment.

Based on the results found, our study revealed that 24\% of the patients
demonstrated a shift in their eligibility for genetic testing when
assessed at a later stage in the oncological process. This leads us to
recommend scheduling a dedicated appointment for the assessment of CPS
in pediatric cancer patients, considering that all the necessary data
may not be available at the time of diagnosis.

The most common reason for changes in the criteria ''pre-data'' vs
''post-data'' is the physical examination and family history. This
discrepancy may occur because certain phenotypic features were not
initially observed at diagnosis or because the patient underwent
physical changes that were not apparent during the initial examination.
A similar situation arises with family history. In some cases, family
history data was inaccurately reported by the families at the time of
diagnosis, or new family members may be subsequently diagnosed with
cancer during the patient's follow-up. The study of CPS should also be
dynamic, and patients who initially do not meet the criteria should be
re-evaluated as new family data becomes available.

In terms of the percentage of patients in whom a pathogenic or likely
pathogenic variant has been identified, our study revealed such variants
in 6\% of patients in our cohort. This percentage is lower than reported
in the literature\textsuperscript{1-5}, most likely attributed to
excluding of patients with specific phenotypes.

Screening all children with cancer is of significant research interest,
however it is accompanied by the trade-off of the cost and reduced
chances of identifying pathogenic variants in patients who do not meet
any risk criteria. This, in turn, presents challenges in managing the
uncertainty associated with VUS. In the context of our research, we have
observed that it is prudent to exclude patients with a strong suspicion
of a particular syndrome. Conducting a WES in such cases may be
unnecessary, as they can be studied more selectively. Moreover, these
selective studies tend to be more cost-effective and are typically
covered by the patient's insurance. For the remaining patients, we have
implemented clinical screening, which considers factors such as family
history, physical characteristics, and, most importantly, the type of
tumor. The type of tumor has been shown to be the most influential
factor in detecting CPS.

Selecting patients by a clinical screening aligns with existing
literature, notable supported by the 2022 review conducted by Rossini et
al\textsuperscript{17}, which discusses various strategies for studying
familial cancer in pediatrics. The authors concluded that the most
efficient approach is to study patients selected using any of the
clinical tools described\textsuperscript{10,13,14,17.}

In terms of determining the most effective genetic approach, whether
WES, trio-WES, a multigene panel, or single gene analysis, there are
limited studies available for comparing their advantages and specific
indications\textsuperscript{8}.

The clinical approach is crucial, and if a specific syndrome is
suspected, a single gene analysis is the preferred option. We recommend
a multigene panel for clinical use due to its comprehensive gene
coverage and targeted information retrieval. WES and whole genome
sequencing should be reserved for research purposes, patients with
unclear cancer or multisystem phenotypes, and those with negative or
inconclusive results from previous single-gene or multigene testing.
Certainly, if resources are available, the most comprehensive
information can be obtained through WES, ideally complemented by pairing
the results with tumor sequencing.

There is some controversy regarding the detection of mutations in genes
associated with the development of cancer in adulthood. In the study by
Sylvester et al\textsuperscript{5}, which compiles the results from six
different cohorts of pediatric patients investigated for the presence of
CPS, various approaches are employed. In all cases, genes related to
adult-onset cancer, such as BRCA2, MSH2 and MSH6, are examined. In the
combined analysis of the 6 cohorts, 0.4\% of patients were found to
carry pathogenic or likely pathogenic variants in BRCA1 and 0.5\% in
BRCA2\textsuperscript{5}. The tumors most frequently associated included
medulloblastoma, neuroblastoma (as in the case of our patient), and
acute lymphoblastic leukemia. Concurrently with the identification of
BRCA2 in our patient, a 30-year-old maternal aunt, that had not been
previously referred to genetic testing, was diagnosed with breast
cancer. Cascade testing was then performed, and the mutation was
identified in the mother, who was informed of the results, leading to
the entire family to undergo genetic counseling.

We advocate for a collaborative approach to the study of CPS, involving
pediatric oncologists and geneticists. The continuity of care through
regular check-ups in pediatric cancer patients, allows for timely
updates on any occurrences of new tumors in the family. Geneticists are
crucial in guiding the approach to cancer predisposition studies and
interpreting genetic tests. Genetic counseling and the long-term
follow-up of patients and their families with CPS should be a
collaborative effort involving pediatric oncologists, geneticists, and
adult oncologists.

\textbf{Conclusions}

The role of the pediatric oncologist in collaboration with geneticists
is fundamental in the study of familial childhood cancer. We recommend
genetic testing by NGS (WES or target panel exome) in all patients if
possible. If it is not possible to test all patients, the care team
should ensure that all patients meeting any risk criteria for having a
CPS are tested. Only in cases of high clinical suspicion of a particular
syndrome, a targeted gene study is an option.

A thorough and accurate family history and a comprehensive physical
examination for syndrome-related data are critical in identifying
patients who are candidates for CPS risk screening. Dedicating a
specific appointment to this topic may increase the likelihood of
identifying these patients. This appointment may be more practical not
at the time of initial diagnosis, but later in the course of the
disease, when the initial concerns of the patient and the family have
been addressed.

\textbf{Declaration of interest statement}

The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of the paper.

\textbf{Acknowledgements}

Almudena Hern\selectlanguage{ngerman}ández, Fátima Peña, Laura Rodriguez, Cristina Almirall

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