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\begin{document}

\title{Lymphangioma of the fetal neck within the PIK3CA-Related-Overgrowth
Spectrum (PROS): A case report.}



\author[1]{Jann Lennard Scharf}%
\author[1]{Michael Gembicki}%
\author[1]{Christoph Dracopoulos}%
\author[1]{Yorck Hellenbroich}%
\author[1]{Anne Offermann}%
\author[1]{Lars Tharun}%
\author[1]{Guido Stichtenoth}%
\author[1]{Kianusch Tafazzoli-Lari}%
\author[1]{Jan Weichert}%
\affil[1]{Universitätsklinikum Schleswig-Holstein - Campus Lübeck}%


\vspace{-1em}



  
  \date{May 08, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Neck tumors are rare fetal conditions. They can receive their growth
stimuli by activating missense mutations characterizing disorders with
benign overgrowth, collectively known as
PIK3CA-Related-Overgrowth-Spectrum. This results in segmental overgrowth
with phenotypic variation, genetic heterogeneity or tissue specific
distribution. Thus, clinical and molecular diagnosis as well as
treatment remains challenging.%
\end{abstract}%



\sloppy


Jann Lennard Scharf\textsuperscript{1} / Michael
Gembicki\textsuperscript{2} / Christoph Dracopoulos\textsuperscript{3} /
Yorck Hellenbroich\textsuperscript{4} / Anne
Offermann\textsuperscript{5} / Guido Stichtenoth\textsuperscript{6} /
Kianusch Tafazzoli-Lari\textsuperscript{7} / Lars
Tharun\textsuperscript{5} / Jan Weichert\textsuperscript{2}

Lymphangioma of the fetal neck within the PIK3CA-Related-Overgrowth
Spectrum (PROS): A case report.

\textsuperscript{1} Department of Gynecology and Obstetrics, University
Hospital Schleswig-Holstein, Campus Lu\selectlanguage{english}beck, Ratzeburger Allee 160, 23538
Lu\selectlanguage{english}beck, Germany, E-mail: JannLennard.Scharf@uksh.de.
https://orcid.org/0000-0003-3685-6997.

\textsuperscript{2~}Department of Prenatal Medicine and Gynecological
Ultrasound, University Hospital Schleswig-Holstein, Campus Lu\selectlanguage{english}beck,
Ratzeburger Allee 160, 23538 Lu\selectlanguage{english}beck, Germany.

\textsuperscript{3} Department of Gynecology and Obstetrics, University
Hospital Schleswig-Holstein, Campus Lu\selectlanguage{english}beck, Ratzeburger Allee 160, 23538
Lu\selectlanguage{english}beck, Germany.

\textsuperscript{4} Institute of Human Genetics, University Hospital
Schleswig-Holstein, Campus Lu\selectlanguage{english}beck, Ratzeburger Allee 160, 23538 Lu\selectlanguage{english}beck,
Germany.

\textsuperscript{5} Institute of Pathology, University Hospital
Schleswig-Holstein, Campus Lu\selectlanguage{english}beck, Ratzeburger Allee 160, 23538 Lu\selectlanguage{english}beck,
Germany.

\textsuperscript{6} Department of Pediatrics, University Hospital
Schleswig-Holstein, Campus Lu\selectlanguage{english}beck, Ratzeburger Allee 160, 23538 Lu\selectlanguage{english}beck,
Germany.

\textsuperscript{7} Department of Pediatric Surgery, University Hospital
Schleswig-Holstein, Campus Lu\selectlanguage{english}beck, Ratzeburger Allee 160, 23538 Lu\selectlanguage{english}beck,
Germany.

\textbf{Abstract}

Neck tumors are rare fetal conditions. They can receive their growth
stimuli by activating missense mutations characterizing disorders with
benign overgrowth, collectively known as
PIK3CA-Related-Overgrowth-Spectrum. This results in segmental overgrowth
with phenotypic variation, genetic heterogeneity or tissue specific
distribution. Thus, clinical and molecular diagnosis as well as
treatment remains challenging.

\textbf{Key Clinicals Message}

The delineation of the prenatal diagnostic key features of PROS
disorders will further assume a crucial part and a prenatal diagnosis of
the causing mutations would provide physicians with a simplified
interdisciplinary perinatal management.

\textbf{Keywords:} PIK3CA, PIK3CA-Related-Overgrowth-Spectrum, PROS,
lymphangioma, lymphatic malformation, cervical, neck, fetal, prenatal,
congenital.

\textbf{Introduction}

Congenital fetal neck tumors constitute exceedingly rare antenatal
conditions. Prenatal diagnosis is usually straightforward by detailed
sonographic examination. According to current literature these lesions
are categorized into different types.\textsuperscript{1--3} The most
frequently described subtypes of neck tumors comprise lymphangiomas,
followed by teratomas and hemangiomas.\textsuperscript{1} However, the
exact incidence of lymphangiomas remains unclear. It considerably varies
whether pre- or postnatal cohorts are analyzed. Prenatal data suggest an
estimated incidence of up to 1 in 1,000 live
births.\textsuperscript{2,4,5}

Although lymphangiomas are non-malignant vascular malformations of the
lymphatic system and histologically mostly benign, extensive and
heterogeneous tumor masses may compress vital cervical structures, so
the final prognosis has been reported to be
poor.\textsuperscript{1,2,6,7}

The clinical course of lymphangiomas is related to the type of lymphatic
malformation and varies from a mild symptomatic that tends to regress
spontaneously to an aggressively invasive growth into surrounding vital
structures.\textsuperscript{5} Spontaneous regression in the latter
cases is unlikely, but possible.\textsuperscript{4,8} By compressing
phenomena, polyhydramnios or airway obstruction can occur, a potentially
life-threatening event.\textsuperscript{5,6,8,9}

Lymphatic malformations like lymphangiomas usually arise from the
defective embryological development of primordial lymphatic
structures.\textsuperscript{3,8,10} Recent data revealed that
lymphangiomas receive their growth stimuli by activating missense
mutations characterizing regional-located tumors in PIK3CA-Related
Overgrowth Spectrum (PROS).\textsuperscript{9,11--13}

PROS encompasses a group of disorders that are predominantly
characterized by benign segmental overgrowth of several tissues with
vascular and lymphatic malformations. Caused by heterozygous, mostly
somatic mosaic-like pathogenic variants in the PIK3CA gene that arise
post-zygotically, affected patients may present with regional-located or
multiple-located findings.\textsuperscript{6,9,14,15} The clinical
picture strongly depends on the embryonic stage in which the causative
mutation occurred, as well as the tissue type affected by this
mutation.\textsuperscript{15} Specific somatic activating mutations in
the phosphatidylinositol-3-kinase/AKT/mTOR (PI3K-AKT-mTOR) pathway lead
to heterogeneous segmental overgrowth phenotypes.\textsuperscript{14}The
PIK3CA gene encodes the 110~kD catalytic alpha subunit (p110\selectlanguage{greek}α\selectlanguage{english}) of the
PI3K protein complex, a lipid kinase of the PI3K-AKT-mTOR pathway, a
signaling pathway, which is crucially involved in the regulation of cell
proliferation, metabolism and survival as well as in angiogenesis.
Hence, PI3K plays a key role in cell growth and division, cell migration
and survival.\textsuperscript{6,9,16} An altered activity of PI3K leads
to uncontrolled cell division and as somatic mosaics, these mutations
may trigger the development of overgrowth syndromes with segmental
growth of several tissues with venous as well as lymphatic malformations
to varying degrees.\textsuperscript{6,9,13,16}

Over the past years, cancer associated PIK3CA mutations have been also
reported to be responsible for a wide range of clinical benign
overgrowth disorders.\textsuperscript{6,17} Recent insights emphasize
the topicality of PROS disorders.\textsuperscript{9,13,18}

Lymphangiomas, developed regionally located in the context of PROS, are
scarcely mentioned in the literature and informative case reports are
almost completely missing. While the acronym PROS is an umbrella term
for various clinical entities, even those entities itself, like
lymphangiomas, can morphologically resemble as a
chameleon.\textsuperscript{14}

In the present report the prenatal and postnatal course of a fetus
suffering from a huge cervical lymphangioma within the PROS will be
delineated. We discuss the clinical picture to what is known from
current literature and focus on the targeted multidisciplinary approach
to establish the final confirmation of an activating missense
mutation-hotspot c.1633G\textgreater{}A;p.Glu545Lys identified by next
generation sequencing.

\textbf{Case presentation}

\textbf{Prenatal course and diagnostics}

A 28-year-old III~G I~P gravida in the 28\textsuperscript{th} week of
pregnancy was referred to our prenatal diagnostic center due to the
suspicion of a fetal neck tumor detected by prenatal ultrasound. The
course of pregnancy was uneventful until the 28\textsuperscript{th} week
of pregnancy. The mother reported one prior spontaneous abortion and had
no regular medication. The parents were of Caucasian origin and
nonconsanguineous and the family history was otherwise unremarkable.

A detailed fetal anatomic ultrasound survey confirmed a fetal neck tumor
with a dimension of 45 x 46 x 39 mm located at the anterior neck,
presenting morphological features of a cystic-caverneous
lymphhemangioma. The tumor extended from the right submandibular region
across the front of the neck to the opposite side, with weak
vascularization. The tumor was supposed to infiltrate the base of the
mouth. Additionally, the mass could not be separated from the upper
thoracic aperture (Fig.~1~a,~b). With the exception of a polyhydramnios,
no further sonomorphological abnormalitiy of the fetus was detected. The
fetus showed an appropriate growth with an estimated fetal weight of
1,020~g corresponded to the 28\textsuperscript{th} percentile. The
parents decided against an amniocentesis. Subsequent ultrasound
examinations within two to three months showed the growth of the tumor
(73 x 68 x 41 mm), which was already strongly vascularized and now
affecting the floor of mouth, but without identifying its entity beyond
any doubt (Fig.~1~c,~d). The parents were interdisciplinary counselled
together with neonatologists and pediatric surgeons. Magnetic resonance
imaging (MRI) was performed, confirming the inhomogeneity as well as the
extent of the tumor but were also unable to clarify the definite tumor
entity.

\textbf{Postnatal neonatological intensive care, diagnostics, therapy
and diagnosis of PROS}

A female infant was delivered weighing 3,450 g
(74\textsuperscript{th}percentile), 49 cm length (27\textsuperscript{th}
percentile) and 35~cm head circumference (71\textsuperscript{th}
percentile) by cesarean section with neonatological standby of an
ex-utero intrapartum treatment (EXIT) procedure in
39\textsuperscript{th} week of gestation (Fig.~2~a,~b). The newborn
adapted well and presented in good condition: APGAR 8/9/10, arterial
umbilical cord pH 7.39. Initially, it required no active management and
was immediately transferred to the neonatologist care unit. During
inpatient stay, the infant underwent an extensive interdisciplinary
neonatological intensive care with regular ultrasound and MRI follow-up
(Fig. 2~c,~d). The lymphangioma seemed to be stable. With intention of
tumor regression as well as obtaining tissue, several sonographically
controlled biopsies of the neck with multiple injections of Picibanil
(OK-432) and the partial resection of areas of the tumor were performed.
Attempting tumor size restriction with Propranolol, based on the
histopathological suspicion of a hemangioma and the meanwhile
significant tumor progression, failed. The histopathological findings
and the clinical presentation of the tumor rather suggested a
lymphangioma. In the meantime, an evaluation by ultrasound and MRIs of
the neck showed a semicircular tumor extension, reaching cranially to
the mastoid and intrathoracally to the carina with increasing
infiltration of the base of the tongue and marked constriction of the
cervical vessels without airway obstruction. With histopathological
evidence of markers of the mTOR pathway, associated with a number of
overgrowth disorders, a therapeutic approach with Sirolimus was
initiated and resulted in a minimal size reduction. Nearly during the
entire inpatient stay and despite an extensive interdisciplinary
management, the etiology of the lympangioma remained unclear and the
conventional molecular genetic investigation a challenging process. To
establish a final diagnosis next generation sequencing (NGS) was
performed and subsequently resulted in the detection of the missense
mutation c.1633G\textgreater{}A;p.Glu545Lys.

\textbf{Discussion}

In certain cases of congenital fetal neck tumors, establishing a
definite prenatal diagnosis might be challenging or even impossible.
Despite all advances in imaging techniques available today it remains
challenging to delineate the underlying cause of such etiologically
heterogeneous tumors in utero. Both, the infant and physicians, as well
as the parents, will benefit from the most accurate clinical and, if
possible, molecular pathological classification of the etiology of the
tumor in order to be able to plan further interdisciplinary treatment.
If the final diagnosis in this case presented had been known earlier, a
number of therapeutic approaches could have been avoided.

During fetal life tumors of the neck tend to impair fetal swallowing,
often resulting in polyhydramnios.\textsuperscript{8} Another serious
thread potentially arises from local compression
phenomena.\textsuperscript{6} Lymphatic malformations of the fetal neck
with lymphangiomas~constitute the most prevalent fetal neck
mass.\textsuperscript{1,2,4,8} In the last two decades there were a
number of attempts to classify these lesions according to their
etiological origin\textsuperscript{8,10}, morphological
appearance\textsuperscript{5,10}, histological
features\textsuperscript{5,7} or postnatal
course.\textsuperscript{10}All these different classifications can
coexist and their characteristics occur side by side. A common and
generally accepted classification of lymphatic malformations is merely
based on histological criteria. There are four distinct histological
types of lymphangiomas consisting of capillary or cavernous
lymphangioma, cystic hygroma and vasculolymphatic
malformation.\textsuperscript{7} However, Eivazi et al. stressed that
this traditional histological classification is obsolete as with
increasing frequency, it has been shown, that anterolateral
lymphangiomas receive their growth stimuli by activating missense
mutations characterizing regional-located tumors in
PROS.\textsuperscript{10--12} Axt-Fliedner et al. mentioned, that
theories of origin of lymphatic malformations include the failure in
connection between the lymphatic and the venous system and that they
could be suitable to explain the origin of lymphangiomas
embryologically.\textsuperscript{5,8} However, they also emphasized,
that in cases with late presenting, regional-located, anterolateral
lymphangiomas, this theory has to be questioned.\textsuperscript{8} It
therefore might be that late-presenting lymphangiomas beyond 30 weeks of
pregnancy rather arise as a result of direct alteration in DNA from a
post-zygotic PIK3CA mutation.

Depending on time of post-zygotic cell division in embryogenesis, either
somatic or, even rarer, constitutional mutations are the
result.\textsuperscript{6,9,15} As one of the most frequent hot-spot
missense mutations, c.1633G\textgreater{}A;p.Glu545Lys was identified in
the helical domain of PIK3CA.\textsuperscript{6,16} Hot spot mutations,
activating, gain-of-function variants, show significantly elevated
biological and biochemical activities.\textsuperscript{14,16} These are
often associated with higher numbers of transformed cell foci suggesting
more rapid cell proliferation.\textsuperscript{17}

In 2015, diagnostic criteria have been defined by Keppler-Noreuil~et~al.
to standardize entities, that originate from PIK3CA mutations, and to
simplify the diagnostic work-up.\textsuperscript{14} Accordingly, in the
present case, the regional-located and sporadic overgrowth of
predominantly lymphatic tissue of the fetal neck, already detected
prenatally and classified as a lymphangioma, combined with the molecular
biological detection of the somatic mutation in the PIK3CA gene with
low-level mosaics, clearly point to the definitive diagnosis of a
disorder within the spectrum of PROS.

Due to their inexorable growth throughout all tissue layers, diagnosis
and treatment of lymphangiomas within PROS disorders is often
difficult.\textsuperscript{3,4,8} However, prenatal ultrasound remains
the primary diagnostic method of choice in the detection of congenital
tumors. Additional volume ultrasound, combined with color Doppler
interrogation, may act as an adjunct in establishing the most likely
diagnosis as it potentially provides valuable information regarding the
spatial relationship to surrounding cervical structures and general
extent of PROS disorders.\textsuperscript{19} The value of an additional
fetal MRI to accomplish the diagnostic work-up and to give further
essential information, particularly in unfavorable maternal conditions
(high Body-Mass-Index) and in complex fetal tumors with multiloculated
masses in a variety of affected anatomical regions has been highlighted
by a number of publications.\textsuperscript{8,19,20} Combined with
information from 3D~volume~ultrasound, it can increase the accuracy of
prenatal diagnosis including location, architecture of tissue, volume or
intracranial or thoracic spread.\textsuperscript{18,19}

Histopathologically, in the present case, the cyst wall-coating cells
presented themselves without expression of D2-40, a lymphatic
endothelial marker, but showed clear CD31 expression, characterizing
blood vascular endothelial cells, so that the finding was classified
most likely as a cavernous hemangioma due to the complex histologic
pattern (Fig.~3). But considering the clinical aspects, a lymphangioma
should be assumed. The D2-40 negativity does not exclude a lymphangioma.
A distinct histological or immunohistochemical differentiation between a
lymphangioma and a hemangioma was not possible. Due to the histological
expression pattern (CD31\textsuperscript{+}/D2-40\textsuperscript{-}),
it is most likely, that endothelial cells harbor the PIK3CA
mutation.\textsuperscript{13,21}

All case reports published so far highlight the complexity of prenatal
diagnosis of somatic mosaic mutations.\textsuperscript{9,18} In 2014,
Keppler-Noreuil et al. also defined testing eligibility criteria for
somatic PIK3CA mutations.\textsuperscript{17} Adequate tissue sampling
is already a challenge postnatally -- and, above all, as a highly
invasive procedure, prenatally.\textsuperscript{17,18} Maybe cultured
amniocytes obtained by amniocentesis for prenatal diagnosis might be
promising and are less invasive.\textsuperscript{9,17,18}

Using NGS, the sensitivity of genetic testing has improved significantly
and molecular diagnosis of somatic overgrowth has become
feasible.\textsuperscript{15--17} By now, mosaic levels can be detected
with a variant allele frequency (VAF) down to
1~\%.\textsuperscript{16}In the tissue received in this case from
multiple biopsies, the activating missense mutation-hotspot
c.1633G\textgreater{}A;p.Glu545Lys with low-grade somatic mosaic and a
VAF of 6.5~\% was detected. This is one of the most frequent mutation
hotspots in the PIK3CA gene, causing somatic overgrowth in PROS. Only
with the change from the pure morphologic description of lymphangiomas
to the recognition of their molecular causes, they can be categorized
into the spectrum of PROS diseases.

The identification of a component of the PI3K-AKT-mTOR signaling pathway
can partly explain the effect of the mTOR-inhibitor Sirolimus, which
resulted temporarily in a minimal size reduction. The targeted
application of a PIK3-inhibitor could be more promising, even as a
serious alternative treatment to surgical debulking. Finally, obtaining
tissue for histopathological and molecular genetic examination to
confirm the definitive diagnosis is mandatory.\textsuperscript{18}

From a therapeutic perspective, debulking procedures were the most
promising approaches yet.\textsuperscript{3--6,8,13} However, in the
future, a shift from surgical debulking to personalized, targeted
pharmacological intervention has been
proposed.\textsuperscript{15,18}Pharmacological therapy approaches are
sclerosing therapy of fetal neck lymphangiomas with Bleomycin or
Picibanil (OK-432), maybe prenatal. In addition, various molecule
inhibitors, targeting different components of the PI3K-AKT-mTOR
signaling pathway, are under clinical investigation: mTOR-inhibitors
like Sirolimus (SRL or Rapamycin) or Everolimus (RAD-001) and recently,
PIK3-inhibitors like Alpelisib (BYL719). Due to the increasing
understanding of molecular biology of PROS disorders, targeted therapies
are highly promising and less invasive. Moreover, it can improve quality
of life of affected patients.\textsuperscript{2,4--6,10,13,22}

Despite all efforts, there is a high risk of
recurrence.\textsuperscript{4,10} Fetal prognosis of congenital tumors
of the neck depends largely on the nature of lesion, on their location
and size, the affected surrounding structures as well as the presence of
other anomalies.\textsuperscript{7,20}

\textbf{Conclusion}

PROS encompasses rare genetic disorders resulting from somatic, mosaic
gain-of-function mutation of the PIK3CA gene. As a result of the genetic
mosaicism, the clinical presentation is extremely variable.
Comprehensive diagnostics are expensive, time-consuming and they place
an unnecessary burden on the infant and its family. A prenatal diagnosis
of this extremely rare mutation enables physicians a simplified
interdisciplinary perinatal management. Moreover, it offers parents and
family members to be involved in the assessment process in the context
of an informed consent. Nevertheless, diagnosis of PROS remains
challenging, both pre- and postnatally and unfortunately, the diagnosis
is rarely made prenatally. Therefore, physicians and parents stay in the
dark for a long time regarding a definitive diagnosis and promising
therapy.

\textbf{Acknowledgments}

Thanks to everyone involved, for their constructive cooperation and
support on this article.

\textbf{Ethical approval statement:} This article does not contain any
studies with human participants performed by any of the authors. The
written informed consent of the patient was given.

\textbf{Author contributions:} All the authors have accepted
responsibility for the entire content of this submitted manuscript and
approved submission. J.L. Scharf: case analysis, literature review,
manuscript writing and editing; J. Weichert: case analysis, literature
review, diagnosis of the patient and patient care, manuscript editing;
M. Gembicki: case analysis, diagnosis of the patient and patient care,
manuscript editing; C. Dracopoulos: manuscript editing; Y. Hellenbroich,
A. Offermann, G. Stichtenoth, K. Tafazzoli-Lari, ; L. Tharun: diagnosis
of the patient and patient care.

\textbf{Research funding:} None declared.

\textbf{Employment or leadership:} None declared.

\textbf{Honorarium:} None declared.

\textbf{Competing interests:} The funding organization(s) played no role
in the study design; in the collection, analysis, and interpretation of
data; in the writing of the report; or in the decision to submit the
report for publication.

\textbf{Conflict of interest:} The authors declare that they have no
conflict of interest.

\textbf{Informed consent:} Informed consent was obtained from the
patient for publication of this case report.

\textbf{References}

1. Kamil D, Tepelmann J, Berg C, et al. Spectrum and outcome of
prenatally diagnosed fetal tumors. \emph{Ultrasound Obstet Gynecol} .
2008;31(3):296-302. doi:10.1002/uog.5260

2. Mikovic Z, Simic R, Egic A, et al. Intrauterine treatment of large
fetal neck lymphangioma with OK-432. \emph{Fetal Diagn Ther} .
2009;26(2):102-106. doi:10.1159/000238111

3. Amodeo I, Colnaghi M, Raffaeli G, et al. The use of sirolimus in the
treatment of giant cystic lymphangioma: Four case reports and update of
medical therapy. \emph{Medicine (Baltimore)} . 2017;96(51):e8871.
doi:10.1097/MD.0000000000008871

4. Knipping S, Bau V. Lymphatische Malformationen im Kopf-Hals-Bereich:
Erfahrungen mit Sklerosierungstherapie. \emph{HNO} . 2011;59(7):683-688.
doi:10.1007/s00106-011-2284-1

5. Grasso D, Pelizzo G, Zocconi E, Schleef J. Lymphangiomas of the head
and neck in children. \emph{Acta Otorhinolaryngol Ital} .
2008;28(1):17-20.

6. Madsen RR, Vanhaesebroeck B, Semple RK. Cancer-Associated PIK3CA
Mutations in Overgrowth Disorders. \emph{Trends Mol Med} .
2018;24(10):856-870. doi:10.1016/j.molmed.2018.08.003

7. Cho JY, Lee YH. Fetal tumors: prenatal ultrasonographic findings and
clinical characteristics. \emph{Ultrasonography} . 2014;33(4):240-251.
doi:10.14366/usg.14019

8. Axt-Fliedner R, Hendrik HJ, Schwaiger C, Ertan AK, Friedrich M,
Schmidt W. Prenatal and perinatal aspects of a giant fetal
cervicothoracal lymphangioma. \emph{Fetal Diagn Ther} . 2002;17(1):3-7.
doi:10.1159/000047996

9. De Graer C, Marangoni M, Romn\selectlanguage{ngerman}ée S, et al. Novel features of
PIK3CA-Related Overgrowth Spectrum: Lesson from an aborted fetus
presenting a de novo constitutional PIK3CA mutation. \emph{Eur J Med
Genet} . 2020;63(4):103775. doi:10.1016/j.ejmg.2019.103775

10. Eivazi B, Werner JA. Lymphatische Malformationen im
Kopf-Hals-Bereich. \emph{HNO} . 2014;62(1):6-11.
doi:10.1007/s00106-013-2803-3

11. Luks VL, N K, Mp V, et al. Lymphatic and other vascular
malformative/overgrowth disorders are caused by somatic mutations in
PIK3CA. \emph{J Pediatr} . 2015;166(4):1048-54.e1.
doi:10.1016/j.jpeds.2014.12.069

12. Rodriguez-Laguna L, Agra N, Ibañez K, et al. Somatic activating
mutations in PIK3CA cause generalized lymphatic anomaly. \emph{J Exp
Med} . 2019;216(2):407-418. doi:10.1084/jem.20181353

13. Le Cras TD, Goines J, Lakes N, et al. Constitutively active PIK3CA
mutations are expressed by lymphatic and vascular endothelial cells in
capillary lymphatic venous malformation. \emph{Angiogenesis} .
2020;23(3):425-442. doi:10.1007/s10456-020-09722-0

14. Keppler-Noreuil KM, Rios JJ, Parker VER, et al. PIK3CA-Related
Overgrowth Spectrum (PROS): Diagnostic and Testing Eligibility Criteria,
Differential Diagnosis, and Evaluation. \emph{Am J Med Genet A} .
2015;0(2):287-295. doi:10.1002/ajmg.a.36836

15. McNulty SN, Evenson MJ, Corliss MM, et al. Diagnostic Utility of
Next-Generation Sequencing for Disorders of Somatic Mosaicism: A
Five-Year Cumulative Cohort. \emph{Am J Hum Genet} .
2019;105(4):734-746. doi:10.1016/j.ajhg.2019.09.002

16. Spier I, Aretz S. Überwuchssyndrome durch Mutationsmosaike im
PI3K-AKT-Signalweg. \emph{medgen} . 2017;29(3):306-313.
doi:10.1007/s11825-017-0153-3

17. Keppler-Noreuil KM, Sapp JC, Lindhurst MJ, et al. Clinical
delineation and natural history of the PIK3CA-related overgrowth
spectrum. \emph{Am J Med Genet A} . 2014;164A(7):1713-1733.
doi:10.1002/ajmg.a.36552

18. Emrick LT, Murphy L, Shamshirsaz AA, et al. Prenatal diagnosis of
CLOVES syndrome confirmed by detection of a mosaic PIK3CA mutation in
cultured amniocytes. \emph{Am J Med Genet A} . 2014;164A(10):2633-2637.
doi:10.1002/ajmg.a.36672

19. Tseng J-J, Chou M-M, Chen W-H. Prenatal 3- and 4-dimensional
Ultrasonographic Findings of Giant Fetal Nuchal Hemangioma.\emph{Journal
of the Chinese Medical Association} . 2007;70(10):460-463.
doi:10.1016/S1726-4901(08)70040-6

20. Knox EM, Muamar B, Thompson PJ, Lander A, Chapman S, Kilby MD. The
use of high resolution magnetic resonance imaging in the prenatal
diagnosis of fetal nuchal tumors. \emph{Ultrasound in Obstetrics \&
Gynecology} . 2005;26(6):672-675. doi:https://doi.org/10.1002/uog.2601

21. North PE. Classification and Pathology of Congenital and Perinatal
Vascular Anomalies of the Head and Neck. \emph{Otolaryngol Clin North
Am} . 2018;51(1):1-39. doi:10.1016/j.otc.2017.09.020

22. Keppler-Noreuil KM, Parker VER, Darling TN, Martinez-Agosto JA.
Somatic Overgrowth Disorders of the PI3K/AKT/mTOR Pathway \& Therapeutic
Strategies. \emph{Am J Med Genet C Semin Med Genet} .
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