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\begin{document}

\title{Identification of an unusual combination of actionable mutations through
genomic profiling in a child with an aggressive sarcoma.}



\author[1]{Shimaa Sherif}%
\author[1]{Wouter R. L. Hendrickx}%
\author[1]{Christophe Raynaud}%
\author[1]{William Mifsud}%
\author[1]{Davide Bedognetti}%
\author[1]{Ata Maaz}%
\affil[1]{Sidra Medicine}%


\vspace{-1em}



  
  \date{May 18, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Rhabdomyosarcoma (RMS) is a highly aggressive pediatric cancer known for
its therapeutic challenges, particularly when it becomes metastatic or
recurrent. Traditional treatment approaches have shown limited success,
prompting a growing interest in targeted therapies. The emergence of
next-generation sequencing (NGS) has revolutionized our ability to
identify actionable mutations in RMS, offering the promise of
personalized treatments and improved patient outcomes. A recent case
involving a 3-year-old child diagnosed with embryonal RMS highlighted
the potential of next-generation sequencing (NGS) in clinical practice.
Despite receiving initial chemotherapy, the patient's tumor showed
progressive growth. What made this case particularly intriguing was the
discovery of co-occurring somatic mutations in the RAS/MAPK pathway,
specifically in BRAF and HRAS genes, which are traditionally believed to
be mutually exclusive. Notably, the BRAF mutation identified in this
case, N581I, is a non-classical (Class III) hotspot mutation that had
not been previously reported in embryonal RMS. This novel finding
underscores the critical importance of comprehensive genetic profiling
in pediatric cancers and suggests the existence of potential therapeutic
avenues that target BRAF alterations. In conclusion, the integration of
NGS technologies in clinical practice holds great promise for
identifying previously unrecognized mutations in pediatric cancers like
RMS. These findings have the potential to open up new treatment options
and improve outcomes for young patients facing this aggressive disease.%
\end{abstract}%



\sloppy


\textbf{Identification of an unusual combination of actionable mutations
through genomic profiling in a child with an aggressive sarcoma.}

Shimaa Sherif, PhD\textsuperscript{1,2}, Wouter R. L. Hendrickx,
PhD\textsuperscript{1,2}, Christophe M. Raynaud, PhD\textsuperscript{1},
William Mifsud MD, PhD\textsuperscript{3}, Davide Bedognetti, MD,
PhD\textsuperscript{1,2*}, Ata Ur Rehman Maaz, MD \textsuperscript{4*}.

\textsuperscript{1}Tumor Biology and Immunology laboratory, Research
Branch, Sidra Medicine, Doha, Qatar

\textsuperscript{2}College of Health and Life Sciences, Hamad Bin
Khalifa University, Qatar Foundation, Doha, Qatar

\textsuperscript{3}Anatomical Pathology, Sidra Medicine, Doha, Qatar

\textsuperscript{4}Pediatric Hematology and Oncology Division, Sidra
Medicine, Doha, Qatar

\textsuperscript{*} Correspondence to:

Ata Maaz, MD, Pediatric Hematology and Oncology Division, Sidra
Medicine, Doha, Qatar, Email: amaaz@sidra.org.

Davide Bedognetti, MD, PhD, Tumor Biology and Immunology laboratory,
Research Branch, Sidra Medicine, Doha, Qatar, Email:
davidebedognetti@gmail.com.

Brief running title: RAS-RAF double mutations in aggressive sarcoma.

Key words: Embryonal Rhabdomyosarcoma, genomic profiling, HRAS, BRAF

Abbreviations\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
ERMS & Embryonal Rhabdomyosarcoma\tabularnewline
NGS & next-generation sequencing\tabularnewline
PCGR & Personal Cancer Genome Reporter\tabularnewline
RMS & Rhabdomyosarcoma\tabularnewline
WES & Whole Exome Sequencing\tabularnewline
WGS & Whole Genome Sequencing\tabularnewline
\bottomrule
\end{longtable}

\subsection*{}

{\label{section}}

\subsection*{ABSTRACT}

{\label{abstract}}

Rhabdomyosarcoma (RMS) is a highly aggressive pediatric cancer known for
its therapeutic challenges, particularly when it becomes metastatic or
recurrent. Traditional treatment approaches have shown limited success,
prompting a growing interest in targeted therapies. The emergence of
next-generation sequencing (NGS) has revolutionized our ability to
identify actionable mutations in RMS, offering the promise of
personalized treatments and improved patient outcomes. A recent case
involving a 3-year-old child diagnosed with embryonal RMS highlighted
the potential of next-generation sequencing (NGS) in clinical practice.
Despite receiving initial chemotherapy, the patient's tumor showed
progressive growth. What made this case particularly intriguing was the
discovery of co-occurring somatic mutations in the RAS/MAPK pathway,
specifically in BRAF and HRAS genes, which are traditionally believed to
be mutually exclusive.

Notably, the BRAF mutation identified in this case, N581I, is a
non-classical (Class III) hotspot mutation that had not been previously
reported in embryonal RMS. This novel finding underscores the critical
importance of comprehensive genetic profiling in pediatric cancers and
suggests the existence of potential therapeutic avenues that target BRAF
alterations.

In conclusion, the integration of NGS technologies in clinical practice
holds great promise for identifying previously unrecognized mutations in
pediatric cancers like RMS. These findings have the potential to open up
new treatment options and improve outcomes for young patients facing
this aggressive disease.

\subsection*{INTRODUCTION}

{\label{introduction}}

Rhabdomyosarcoma (RMS) is an aggressive pediatric cancer originating
from embryonic mesenchymal tissue\textsuperscript{1}, and remains a
formidable therapeutic challenge due to its ability to manifest in
diverse anatomical sites\textsuperscript{2}. Despite conventional
treatments, including chemotherapy, surgery, and radiotherapy, the
overall survival rate for metastatic and recurrent RMS patients remains
low. This bleak scenario has propelled interest in exploring targeted
therapies, particularly in high-risk patients and those with aggressive
and relapsed disease. Recent advancements in next-generation sequencing
(NGS) and computational analyses have revolutionized the study of
somatic mutations across various cancer types, shedding light on novel
opportunities for precision medicine in RMS and beyond.

Sidra Medicine, the single pediatric cancer care institution in Qatar,
has initiated the Sidra Pediatric Precision Oncology Program, aiming to
provide research-grade tumor sequencing for pediatric solid tumor
patients. This initiative offers an unparalleled avenue for molecular
characterization that guides both diagnosis and treatment.

In this context, we present a case report of a young patient with
embryonal RMS, treated at Sidra Medicine. In our patient, we identified
the co-occurrence of two actionable mutations in the HRAS and BRAF genes
part of the RAS/MAPK pathway. These mutations are traditionally observed
as mutually exclusive, and their combination has never been reported (to
our knowledge) in embryonal RMS.

\subsection*{METHODS}

{\label{methods}}

Informed written consent was obtained from the patient for participation
in the study (SDR SDR200074 / IRB 1509000). The institutional review
board approved this study (conducted at Sidra Medicine). At
presentation, his tumor was diagnosed with the standard
histopathological examination at the referring center. At the time of
progression, further analysis was carried out on the tumor DNA extracted
from the FFPE specimen by next-generation sequencing (NGS) technique.

\textbf{DNA extraction~}

Blood:

Blood was diluted with 1 volume of DPBS (\#14190, Gibbco). Sepmate tube
were filled with 4ml of Lymphoprep, and diluted blood was added on top.
PBMCs were separated with 10 minutes of centrifugation at 1200g. The
supernatant was collected, and cells were further diluted in 10 mL of
additional DPBS. After centrifugation of 8 minutes at 400g, the
supernatant was discarded, and cells were resuspended in 1ml of DPBS for
another 8 minutes of centrifugation at 400g. Cells were resuspended in
200 \selectlanguage{greek}μ\selectlanguage{english}L of DPBS with 20 \selectlanguage{greek}μ\selectlanguage{english}L of Proteinase K and 200 \selectlanguage{greek}μ\selectlanguage{english}L of Buffer AL. After
vertexing, the mix was incubated for 10 minutes at 56C. 200 \selectlanguage{greek}μ\selectlanguage{english}L of
ethanol (\#459836, Sigma-Aldrich) was added to the mix and mixed by
pipetting before being transferred to the DNeasy minispin column. DNA
was loaded on the column by centrifugation 1 min at 6000g. DNA was
purified with 500 \selectlanguage{greek}μ\selectlanguage{english}L of AW1 buffer and centrifuged at 6000g for 1
minute. Then washed with 500 \selectlanguage{greek}μ\selectlanguage{english}L of AW2 buffer then centrifuged at 20000g
for 3 minutes.

Tissue samples:

Samples were thawed and resuspended in 600\selectlanguage{greek}μ\selectlanguage{english}L of RLT Plus buffer. Samples
were homogenized with a syringe with a 20-gauge needle. The lysate was
centrifuged at 20000g for 3 min and the supernatant was transferred to
the AllPrep DNA spin column and centrifuged at 8000g for 1 minute. DNA
was further purified by the addition of 500\selectlanguage{greek}μ\selectlanguage{english}L of AW1 buffer and
centrifuged at 8000g for 1 minute, followed by 500\selectlanguage{greek}μ\selectlanguage{english}L of AW2 buffer and
centrifugation at 20000g for 2 minutes.

Whole Exome Sequencing (WES)

Libraries were sequenced on Illumina's HiSeq 4000 platform using 150 bp
paired-end reads (150PE) at the Genomics Core, Sidra Medicine. Reads
were mapped to reference genome hs37d5 (1000 genomes Phase2 Reference
Genome Sequence) based on GRCh37/hg19 using BWA
(v.0.7.12)\textsuperscript{3}.

Single-nucleotide variants (SNVs) were called using mutect
(v.1.1.7)\textsuperscript{4} and somatic small insertions and deletions
(indels) using strelka2\textsuperscript{5} (bcbio-nextgen
v.1.1.1)\textsuperscript{6}. To filter out false-positive SNP calls, the
fpfilter\textsuperscript{7} was used, and the applied filtering
parameters are specified in the fpfiler.pl script shared on GitHub.
Subsequently, Mutation Annotation Format (MAF) files were generated
using the VCFtoMAF tool (v.1.6.16)\textsuperscript{8} which also
appended the SIFT, PolyPhen and ExAc annotations. The~Personal Cancer
Genome Reporter~(PCGR)\textsuperscript{9} was created from the VCF files
and delivered to the oncologists.

\textbf{Clinical validation}

Paraffin block was sent for clinical validation in a clinically
accredited laboratory, the DNA extraction was performed using the Qiagen
Epitect FFPE Lysis Kit. Mutation analysis was performed using
next-generation sequencing ion torrent technology (the method has an
evaluation sensitivity of mutations of at least 5\%).

\subsection*{RESULTS}

{\label{results}}

\textbf{Case representation}

A 3-year-old male child was referred with a right-sided neck swelling.
Anatomical imaging with an MRI scan at the referring center demonstrated
a mass in the right masticator space measuring 3.3.x2.7x3.2 cm involving
the medial and lateral pterygoid muscles. Review of histological
material received from the referring center, obtained from an incisional
biopsy of this mass, showed primitive mesenchymal cells with loose,
myxoid stroma, and variable degrees of skeletal muscle differentiation.
Immunohistochemistry showed strong and diffuse Desmin expression,
nuclear expression of Myogenin in fewer than 50\% of tumor cells, and
nuclear expression of MyoD1 in approximately 50\% of tumour cells
(Figure 1). The histological pattern was consistent with embryonal RMS.
There was no evidence of \emph{FOXO1} rearrangement by
fluorescent\emph{in-situ} hybridization (FISH). Stage evaluation with
PET/CT and bone marrow examination was negative for metastatic disease.
The patient was started on chemotherapy according to the 'intermediate
risk' guidelines of the institutional protocol. After the initial 9
weeks of chemotherapy, response evaluation demonstrated tumor
progression on MR and PET/CT imaging. At this point, his chemotherapy
was intensified according to the 'high-risk' guidelines. Since the tumor
was surgically not amenable to resection, he received focal radiotherapy
(59.4Gy) for local control, followed by consolidative adjuvant
chemotherapy according to the high-risk protocol. He was then treated
with maintenance therapy for 12 months. At the end of his chemotherapy,
the tumor mass was stable on MRI and demonstrated a complete metabolic
response on PET/CT. However, the risk of progression/relapse is
extremely high.

\textbf{Identification of dual RAS pathway mutations}

Using Whole Exome Sequencing of tumor tissue and blood, we identified
several somatic mutations in this patient (Table 1). Of these mutations
is the c.38 G\textgreater{}T(p.Gly13Val) hotspot mutation in the HRAS
gene (Supplementary Figure 1). HRAS is a GTPase Proto-Oncogene that is
located on chromosome 11(11p15.5). GTP-bound ``on'' state Ras proteins
convert extracellular stimuli into intracellular signaling cascades,
which eventually produce changes in cellular functions; this signaling
stops when Ras-bound GTP is hydrolyzed to GDP as a consequence of
another signaling cascade. Thus, in normal cells, Ras proteins function
as molecular regulators for critical changes in cellular activities,
such as cell proliferation and survival, and their appropriate
regulation is crucial to maintaining the homeostasis of cells and,
eventually, the entire organism. While in case of mutated RAS gene,
especially in the codons 12, 13, and 61, results in reduced intrinsic
GTPase activity, that in turn leads to permanent activation of RAS genes
and downstream signaling pathways, thereby mediating malignant
transformation\textsuperscript{10,11}.

In addition to the HRAS mutation, we identified another mutation in the
same pathway which is the c.1742 A\textgreater{}T (p.Asn581Ile) in the
BRAF gene. BRAF represents one of the most frequently mutated protein
kinase genes in the human tumors\textsuperscript{12}. BRAF gene encodes
Serine/Threonine Kinase and is located on~chromosome 7(7q34). There are
three classes of BRAF mutations that were identified, they are grouped
based on the kinase activity,~dependence on RAS, and dimerization.

Class 1 consists of BRAF V600 mutants, and classes II and III are
non-V600 BRAF mutations. In our patient, we identified
c.1742A\textgreater{}T, a missense class III BRAF mutation in addition
to the c.1742 A\textgreater{}T mutation in the HRAS gene, both are
deleterious hotspot mutations involved in the RAS signaling pathway
(Table 1).

The IGV coverage for the BRAF and HRAS mutations detected in our patient
is 44.8\% and 52\% respectively with 0 detections in the blood that
confirm being somatic mutations (Supplementary Figure 2).

Clinical validation

The sample was sent for clinical validation. The outcome of the
molecular examination confirmed the presence of the mutation c.3551
G\textgreater{} A (p. Gly1184Glu) in the codon 1184 of exon 23 of
the\textbf{ALK} gene with a frequency of 16.06\%. The presence of the
mutation c.1621 A\textgreater{} C (p. Met541Leu) in codon 541 of exon 10
of the \textbf{KIT} gene with a frequency of 50.23\%. The presence of
the mutation c.1416 A\textgreater{} T (p. Gln472His) in codon 472 of
exon 11 of the \textbf{KDR} gene with a frequency of 60.22\%. The
presence of the mutation c.1742 A\textgreater{} T (p. Asn581Ile) in
codon 581 of exon 15 of the BRAF gene with a frequency of 41.13\%. The
presence of the canonical mutation c.2651 A\textgreater{} T
(p.Glu884Val) in codon 884 of exon 15 of the \textbf{RET} gene with the
frequency of 11.43\%. The presence of the canonical mutation c.38
G\textgreater{} T (p. Gly13Val) in codon 13 of exon 2 of
the\textbf{HRAS} gene with a frequency of 50.35\%. The presence of the
canonical mutation c.767 A\textgreater{} T (p.Gln256Leu) in codon 256 of
exon6 of the \textbf{SMAD4} gene. And the presence of the canonical
mutation c.215 C\textgreater{} A (p. Thr72Lys) in codon 72 of exon 2 of
the \textbf{SMARCB1} gene.

The number of mapped Reads is 379,632 reads and the mean depth is 1,491,
On target is 97.40\%.

\textbf{Class III BRAF mutations highly occurred with RAS mutations.}

We analyzed publicly available somatic mutation data from 65853
patients~across different cancer types (http://cbioportal.org) (Table 2,
Supplemental Figure S3). We found only 1606 patients with class I BRAF
mutations, around 95\% of them bearing BRAF V600E mutations and 4\%
co-expressed RAS mutations with frequencies 0.37\%, 2.5\% and 0.8\% for
HRAS, NRAS and KRAS respectively. In addition, we identified 289
patients with class II BRAF mutations. Of them; \textasciitilde{}17\%
co-expressed RAS mutations with frequencies 2.77\%, 3.8\% and 10.38\%
for HRAS, NRAS and KRAS respectively. Conversely, 286 patients with
class III BRAF mutations were found, 24\% of them co-expressed RAS
mutations with frequencies 3.85\%, 8.74\% and 11.5\% for HRAS, NRAS and
KRAS respectively. The class III mutation N581I that was detected in our
patients co-occurred with RAS mutations in around third of the cases
(33\%). Of these cases 6.67\%, 6.67\% and 20\% co-occurred with HRAS,
NRAS and KRAS respectively.

We investigated the frequency of the three BRAF mutation classes in
sarcomas, the overall number of sarcoma patients with BRAF mutations is
25 patients, of them; five patients with class I BRAF mutation (0, 0 and
1 co-occurred with HRAS, NRAS and KRAS respectively), four with class II
BRAF mutations (0, 1 and 0 co-occurred with HRAS, NRAS and KRAS
respectively), and two patients with class III mutations (0, 0 and 0
co-occurred with HRAS, NRAS and KRAS respectively). While the remaining
are with other mutation types including intergenic and fusion BRAF
genes.

Looking at the overall occurrence of the BRAF mutations in RMS, we
identified two RMS patients with class I, class II mutations across all
the datasets and none of them co-occurred with HRAS mutations.
Highlighting the very rare occurrence of BRAF mutations in RMS.

\textbf{DISCUSSION}

The RAS-RAF-MEK-ERK cascade (RAS-RAF mitogen-activated protein kinase;
MAPK) is the key regulatory pathway for cell growth, proliferation,
differentiation, and apoptosis. Signaling through this pathway typically
occurs through different plasma membrane growth factor receptors that
trigger RAS family GTPases, including HRAS, NRAS and KRAS. Activated RAS
proteins can recruit members of the RAF kinase family (ARAF, BRAF, and
CRAF) to the plasma membrane, complex with and activate them. Activated
RAF kinases signal downstream to other MEK/ERK cascade elements to over
150 downstream targets, nuclear and cytosolic. The MAPK pathway is
frequently dysregulated in cancer, often via mutations of its
intracellular components or activation of growth factor receptor
tyrosine kinases. BRAF is the most potent form of RAF
kinases\textsuperscript{13}. T1799A transversion mutation in BRAF
(BRAFv600) accounts for more than 80\% of all known BRAF mutations and
leads to hyperactivation of the MAPK pathway\textsuperscript{12,14} that
results in deregulated downstream signaling and consequently,
unregulated cell proliferation and survival, which contributes to
oncogenesis. Patients harboring BRAF v600 mutations are treated by BRAF
inhibitors, such as vemurafenib, dabrafenib and encorafenib. However,
other BRAF mutations like Class III BRAF mutations were recently
identified as being resistant to these agents.

In our patient, we identified two somatic mutations in the RAS pathway
genes (BRAF N581I and HRAS G13V), which are mutually
exclusive\textsuperscript{15}. BRAF N581I is a Class III hotspot
mutation that lies within the protein kinase domain of the Braf protein
(UniProt.org). Class III BRAF mutants display low kinase activity or are
kinase-dead. They require the co-existence of other upstream mutations
to give the oncogenic signaling. They trigger the MAPK pathway through
enhanced RAS binding and subsequent RAS-dependent CRAF activation
resulting in elevated ERK signaling and are susceptible to ERK-mediated
feedback\textsuperscript{16--18}. (Supplementary Figure 4).

One study reported the cooperation of the BRAF and HRAS in histiocytic
sarcoma. In this study, the BRAF mutation Phe595Leu (class III)
co-occurred with HRAS (Q61R). The co-occurrence of these two mutations
causes a higher oncogenic signal than in the case of BRAF
alone\textsuperscript{19}. This study also highlighted the efficacy of
Pan-RAF inhibitors, such as sorafenib and AZ628, and the MEK inhibitor
trametinib in interfering with the MEK/ERK phosphorylation driven by the
cooperative activity of BRAF (F595L) and oncogenic RAS. Another study
introduced the KIN-2787 as a potent and selective small-molecule pan-RAF
inhibitor specifically designed to inhibit Class II and III BRAF
dimers\textsuperscript{18}.

Class III mutations are sensitive to the inhibition of RAS activation by
inhibitors of receptor tyrosine kinases. A multicenter analysis
suggested that anti-EGFR therapy has promising effects for some patients
with metastatic colorectal cancer that is not v600 BRAF mutated. The
study demonstrated that half of the patients with kinase-impaired and
RAS-dependent BRAF mutations responded to the therapy, whereas almost
none of those with kinase-activating and RAS-independent mutations
did\textsuperscript{20}. In contrast, current RAF inhibitors are not
expected to effectively inhibit the ERK pathway in these tumors. It was
reported that RAF inhibitor vemurafenib failed to inhibit ERK signaling
in tumor cells that express class III BRAF mutants. However, cell lines
harboring Class III mutations are sensitive to classic RAS/MAPK singling
inhibitors \textsuperscript{21}.

\textbf{CONCLUSION}

The elucidation of complex mutation profiles through NGS signifies a
paradigm shift in oncological research. Unveiling the co-occurrence of
BRAF and HRAS mutations in RMS, especially the enigmatic Class III BRAF
mutation, holds the potential to redefine therapeutic strategies.
Targeting these unique mutations presents a remarkable opportunity for
innovative treatment modalities.

\textbf{FIGURES AND TABLES LEGEND}

Figure1. Histology

\begin{enumerate}
\tightlist
\item
  H\&E-stained section showing cells with a mesenchymal appearance set
  in a loose background stroma, and showing with variable (in this
  focus, relatively little) skeletal muscle differentiation.
\item
  Desmin immunohistochemistry, showing diffuse cytoplasmic expression in
  neoplastic cells
\item
  Myogenin immunohistochemistry, showing nuclear expression in fewer
  than 50\% of neoplastic cells
\item
  MyoD1 immunohistochemistry, showing nuclear expression in
  approximately 50\% of neoplastic cells
\end{enumerate}

Table1. Somatic mutation detected by Whole-exome sequencing.

Table2. Frequency of BRAF/RAS mutations co-occurrence

\textbf{SUPPORTING INFORMATION}

Supplemental Figure S1. The HRAS and BRAF detected mutations in the RMS
patient.

Supplemental Figure S2. IGV of the detected mutations.

Supplemental Figure S3. Percent frequency of co-occurrence of HRAS
mutations with BRAF classes I, II and III in cBioportal.

Supplemental Figure S4. Diagram of Class I, II and III BRAF mutations
and the possible CLASS III inhibitors.

\subsection*{CONFLICT OF INTEREST}

{\label{conflict-of-interest}}

The authors declare that there is no conflict of interest.

\subsection*{ACKNOWLEDGMENTS}

{\label{acknowledgments}}

Funding This work was supported by grants from Qatar National Research
Fund (QNRF grant

PPM 05-0316-210001)

\subsection*{AUTHORS CONTRIBUTION}

{\label{authors-contribution}}

S.S. Processed the WES data, collected the public data, performed the
bioinformatic analysis and drafted the manuscript. W.H. Supervised the
WES bioinformatic processing pipelines. C.R. extracted the DNA. A.M.
Collected the clinical data. C.R. W.H., D.B., A.M., and W.M.
participated in the interpretation of the results.

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doi:10.1200/JCO.2021.39.15\_suppl.3116\selectlanguage{english}
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\textbf{Hosted file}

\verb`Table 2. Frequency of BRAF_RAS mutations co-occurrence.xlsx`  available at \url{https://authorea.com/users/666273/articles/667201-identification-of-an-unusual-combination-of-actionable-mutations-through-genomic-profiling-in-a-child-with-an-aggressive-sarcoma}

\textbf{Hosted file}

\verb`Table1. Somatic mutation detected by Whole exome sequencing.xlsx`  available at \url{https://authorea.com/users/666273/articles/667201-identification-of-an-unusual-combination-of-actionable-mutations-through-genomic-profiling-in-a-child-with-an-aggressive-sarcoma}

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