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\begin{document}

\title{Targeted Therapy with Vemurafenib in Brazilian Children with Refractory
Langerhans Cell Histiocytosis: Two Case Reports and a Comprehensive
Review}



\author[1]{Klerize Anecely de Souza Silva}%
\author[2]{Isis Maria Quezado Soares Magalhães}%
\author[3]{Daniela Elaine Roth Benincasa}%
\author[1]{Daiane Keller Cecconello}%
\author[1]{Mariana Bohns Michalowski}%
\affil[1]{Universidade Federal do Rio Grande do Sul}%
\affil[2]{Hospital da Crianca de Brasilia Jose Alencar}%
\affil[3]{Hospital Conceicao}%


\vspace{-1em}



  
  \date{June 08, 2024}


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\selectlanguage{english}
\begin{abstract}
Langerhans cell histiocytosis (LCH) is characterized by inflammatory
lesions featuring clonal infiltration. The BRAF mutation stands out as
the most prevalent. Vemurafenib has emerged as a therapeutic option. The
off-label use of VMF persists in Brazil. We will delineate the cases of
two Brazilian children suffering from LCH with a positive response to
VMF. Case Report I: The patient underwent standard therapy, but bone
marrow infiltration persisted. After confirming a BRAF V600E mutation
through testing, VMF was initiated, resulting in significant
improvements. Case Report II: The patient initiated VMF and showed a
positive response. After several months, maintenance therapy was
introduced. However, skin lesions recurred upon discontinuation of VMF.
She has been on VMF for a total of 31 months, maintaining remission. The
cases presented represent the first reported instances of off-label VMF
use in Brazil for the treatment of LCH, and both patients have
demonstrated excellent responses to the medication.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Langerhans cell histiocytosis (LCH) is an exceptionally rare malignancy
characterized by inflammatory lesions featuring pathological clonal
infiltration of cells belonging to the mononuclear phagocyte system,
which exhibit phenotypical traits resembling Langerhans cells, notably
expressing CD1a and CD207 (1). It is an exceptionally rare disease, with
reported incidence rates ranging from 2.6 to 8.9 cases per million among
children under 15 years of age, peaking at an average age of
approximately three years (2). Notably, the disease presents a diverse
clinical course, ranging from self-limited forms to aggressively
progressive multisystemic variants, often entailing an unfavorable
prognosis (3).

To address this diversity, the therapeutic approach to LCH is
intricately tailored based on the extent of organ involvement. In cases
of localized, single-organ disease, a watch-and-wait strategy may
suffice. Alternatively, when isolated bone involvement occurs, surgical
interventions may be considered. Notably, localized disease often
carries a favorable prognosis (4).

Conversely, multisystemic disease is characterized by the presence of
affected risk organs such as the bone marrow, spleen, and liver,
significantly worsening the prognosis (5). One persistent challenge in
LCH treatment is the notable rate of disease reactivation, particularly
in instances where risk organs are implicated (6). Patients with
refractory histiocytosis typically exhibit characteristics such as age
under 2 years, risk organ involvement, elevated inflammatory markers,
resistance to standard treatment, and often harbor the BRAF V600E
mutation (7).

Recent advancements have illuminated the pathophysiology of LCH,
unveiling the pivotal role of the ''Ras/Raf/MEK/ERK'' signaling pathway
in myeloid differentiation. Within the spectrum of mutations, the BRAF
mutation stands out as the most prevalent, accounting for over 50\% of
LCH cases. It is closely associated with severe clinical presentations,
heightened resistance to conventional chemotherapy, and an increased
risk of relapse (8). The BRAF gene regulates the synthesis of the BRAF
protein, a crucial component of the RAS/MAPK signaling pathway that
governs fundamental cellular processes such as proliferation,
differentiation, migration, and apoptosis (9). In the presence of BRAF
mutations, the aberrant BRAF protein perpetuates unregulated signaling
to the cell nucleus, fueling unrestrained growth and differentiation of
Langerhans cells (10). Other genetic mutations, including those within
the MAP2K and ARAF genes, have also been identified (11).

With these recent molecular insights into the central role of the BRAF
mutation in LCH pathogenesis, Vemurafenib has emerged as a therapeutic
option for children grappling with high-risk multisystemic disease that
proves refractory to conventional treatments or experiences recurrent
relapses. Vemurafenib (VMF), initially approved for metastatic melanoma
in adults, has demonstrated the capacity to target and inhibit the BRAF
V600E mutation (12). While the \emph{off-label} use of VMF in refractory
multisystemic LCH among children persists in Brazil, the European
Medicines Agency has sanctioned its utilization. Observational studies
in Europe have already attested to the safety and efficacy of VMF in
children afflicted with refractory LCH carrying the BRAF V600E mutation
(13). However, it is crucial to underscore that this therapeutic
approach has yet to definitively eradicate the neoplastic clone, and
long-term toxicities remain a subject of ongoing investigation (14).

In this paper, we will delineate the cases of two Brazilian children
suffering from refractory LCH, which remained unresponsive to both
standard and second-line treatments but exhibited a positive response to
Vemurafenib therapy. Furthermore, we will provide an extensive review of
the current literature on this subject.

\textbf{CASE REPORT I}

The patient is a 1-year and 9-month-old female child who presented with
pancytopenia and a history of atopic and seborrheic dermatitis, repeated
febrile otitis, impetigo on the scalp, extensive skin lesions on the
perineum, and difficulty walking for 3 months. Initially suspected of
leukemia due to pancytopenia, further examination revealed pale skin,
lichenized skin lesions on the neck, erythematous and moist skin lesions
on various body parts, including the perineum, as well as
hepatosplenomegaly and suppurative otitis.

CT scans showed numerous osteolytic lesions affecting multiple bones,
including the skullcap, skull base, maxilla, mandible, scapulae, ribs,
vertebrae, pelvis, and proximal femurs, in addition to
hepatosplenomegaly. Skin biopsy, bone marrow biopsy, and cerebrospinal
fluid collection confirmed the diagnosis of Multisystem Histiocytosis
(MS-LCH) with involvement of risk organs (liver, spleen, and bone
marrow) and bone structures.

The patient initially received vinblastine and prednisolone but
exhibited refractory disease. Second-line therapy with Cladribine and
Cytarabine also did not eliminate bone marrow infiltration. After a BRAF
V600E mutation test confirmed the mutation, treatment with Vemurafenib
was initiated, leading to significant improvements in bone marrow
infiltration and skin lesions. The patient currently takes Vemurafenib
without significant side effects, maintaining an excellent quality of
life at 4 years and 11 months of age.

\textbf{CASE REPORT II}

The patient is a female child, 1 year and 1 month old, with a history of
recurrent otorrhea, hospitalizations for otitis, diarrhea, and
dehydration. During a hospitalization for inappetence and diarrhea,
hepatosplenomegaly was observed, leading to a referral to a pediatric
oncology service. Physical examination revealed hepatosplenomegaly, a
tumor in the occipital region, skin abnormalities including petechiae on
the thighs and inguinal region, and dermatitis on the scalp.

Imaging confirmed an expansive bone lesion in the occipital region and
hepatosplenomegaly. Biopsies and tests led to a diagnosis of Langerhans
Cell Histiocytosis (LCH). Chemotherapy was initiated according to the
LCH 2009 protocol, with an initial partial response. However, recurrent
hospitalizations were required due to complications. Second-line therapy
with Cytarabine and Cladribine was attempted but had to be suspended due
to septic shock.

A BRAF mutation test confirmed the pV600E mutation in the BRAF gene,
leading to the initiation of Vemurafenib therapy. Initially, Vemurafenib
showed a good response, with a reduction in organomegaly and resolution
of skin lesions. After 12 months of continuous Vemurafenib therapy,
maintenance antineoplastic therapy was introduced according to the LCH
2009 protocol. However, skin lesions recurred after discontinuing
Vemurafenib. The medication was reintroduced, and the patient returned
to the previous remission status. She has been on Vemurafenib for a
total of 31 months, maintaining complete remission, excellent overall
health, and quality of life.

The patient is currently 4 years old and continues to use Vemurafenib as
the sole treatment.

\textbf{DISCUSSION}

The cases presented here represent the first reported instances
of\emph{off-label} Vemurafenib use in Brazil for the treatment of
high-risk histiocytosis refractory to first and second-line therapies.
The discovery of the recurrent BRAF gene mutation's involvement in
histiocytosis pathogenesis is relatively recent. In 2010, Badalian-Very
et al. first described this mutation, suggesting that histiocytosis is a
neoplastic disease driven by the BRAF V600E gain-of-function mutation.
This mutation is found in approximately 57\% of histiocytosis cases and
is a key discovery given the disease's heterogeneity (15). Importantly,
this mutation is not unique to histiocytosis; it is a common oncogenic
mutation observed in about 30 different neoplasms, leading to activation
of the Ras/Raf/MEK/ERK signaling pathway (16). Mutations in MAP2K1 have
been found in 50\% of BRAF wild-type patients, indicating the critical
role of the MAPK pathway in LCH pathogenesis (17). Other mutations
affecting ARAF, MAP3K1, NRAS, PI3CA, and other targets are found in the
remaining 25\% of patients (18).

These molecular findings suggest that LCH can be characterized as a
clonal myeloid neoplasm driven by activation of the Ras/Raf/MEK/ERK
pathway with inflammatory properties. While the exact pathophysiology
remains unclear, this information has paved the way for more
individualized treatments for LCH (14).

In this context, targeted therapy has opened new possibilities for
treating various neoplasms, including histiocytosis. In the case of
histiocytosis, understanding its pathogenesis with the discovery of
recurrent BRAF mutations has led to the use of the selective BRAF
inhibitor, Vemurafenib (19). Initial reports, including the case by
Heritier et al., demonstrated successful use of VMF in children with
resistant MS-LCH (20). Most studies and case reports describe VMF use in
relapsed or refractory histiocytosis cases that did not respond to first
and/or second-line treatment. These cases often involve severely ill
infants who showed rapid responses to VMF, with resolution of symptoms
and transfusion independence. VMF has proven to be a safe treatment with
no serious adverse effects during its use (21).

Prior to targeted therapy, second-line treatments for histiocytosis were
associated with high toxicity, making them less favorable (22).
Hematopoietic stem cell transplantation (HSCT) was considered a rescue
option, but its toxicity was even higher (23).

However, a major concern with long-term VMF treatment is its potential
for toxicity, as observed in adult patients with Erdheim-Chester disease
(24). Fortunately, VMF's safety profile appears more favorable in
infants and children, although long-term effects remain unknown (14,25).

Despite these promising developments, many questions remain unanswered.
When is the right time to start VMF? What is the ideal duration of VMF
treatment? What is the optimal maintenance therapy after stopping VMF?
How long can children safely use VMF? Unfortunately, we lack sufficient
data to answer these questions comprehensively. Additionally, Brazil
lacks epidemiological data on histiocytosis and studies evaluating the
budgetary impact of incorporating VMF into the public health care system
(SUS).

While the expense associated with VMF treatment can be significant, it's
essential to consider that this is a rare disease that primarily affects
young children. Historically, high-risk refractory cases have had low
survival rates, making remission-inducing treatment invaluable. Targeted
therapy has the potential to extend and enhance the quality of life for
patients and their families. These factors should not be underestimated,
as they encompass the reintegration of the child and their family into
society. Nevertheless, it's crucial to acknowledge that further data is
required to firmly solidify these conclusions.

These two cases represent the first instances of
\emph{off-label}Vemurafenib use in Brazil, and both cases have shown
excellent responses to the medication. However, the long-term safety and
efficacy of VMF in children with histiocytosis require further
investigation. Prospective studies are essential to address critical
aspects, including histiocytosis pathophysiology, the sufficiency of
Vemurafenib as a standalone treatment in cases with BRAF mutations, and
the consideration of targeted therapy as a chronic treatment option.

\textbf{REFERENCES}

1. Jezierska M, Stefanowicz J, Romanowicz G, Kosiak W, Lange M.
Langerhans cell histiocytosis in children - a disease with many faces.
Recent advances in pathogenesis, diagnostic examinations and treatment.
Postepy Dermatol Alergol. fevereiro de 2018;35(1):6--17.

2. Haroche J, Cohen-Aubart F, Rollins BJ, Donadieu J, Charlotte F,
Idbaih A, et al. Histiocytoses: emerging neoplasia behind inflammation.
Lancet Oncol. fevereiro de 2017;18(2):e113--25.

3. Egeler RM, Katewa S, Leenen PJM, Beverley P, Collin M, Ginhoux F, et
al. Langerhans cell histiocytosis is a neoplasm and consequently its
recurrence is a relapse: In memory of Bob Arceci. Pediatr Blood Cancer.
outubro de 2016;63(10):1704--12.

4. Tzotzola V, Petrikkos L, Papadakis V, Mitropoulou G, Kelaidi C,
Dimitriadis E, et al. Long-term outcome, clinical course and treatment
approaches of paediatric langerhans cell histiocytosis: A greek
reference centre report. Acta Paediatr Oslo Nor 1992. junho de
2021;110(6):1944--51.

5. Tuysuz G, Yildiz I, Ozdemir N, Adaletli I, Kurugoglu S, Apak H, et
al. Langerhans Cell Histiocytosis: Single Center Experience of 25 Years.
Mediterr J Hematol Infect Dis. 2019;11(1):e2019035.

6. Gadner H, Minkov M, Grois N, P\selectlanguage{ngerman}ötschger U, Thiem E, Aricò M, et al.
Therapy prolongation improves outcome in multisystem Langerhans cell
histiocytosis. Blood. 20 de junho de 2013;121(25):5006--14.

7. Bhatia P, Singh M, Sharma M, Sharma A, Kakkar N, Radhika S, et al.
BRAF V600E mutation in childhood Langerhans cell histiocytosis
correlates with multisystem disease and poor survival. Blood Cells Mol
Dis. maio de 2020;82:102356.

8. Héritier S, Hélias-Rodzewicz Z, Lapillonne H, Terrones N, Garrigou S,
Normand C, et al. Circulating cell-free BRAF(V600E) as a biomarker in
children with Langerhans cell histiocytosis. Br J Haematol. agosto de
2017;178(3):457--67.

9. Chakraborty R, Hampton OA, Shen X, Simko SJ, Shih A, Abhyankar H, et
al. Mutually exclusive recurrent somatic mutations in MAP2K1 and BRAF
support a central role for ERK activation in LCH pathogenesis. Blood. 6
de novembro de 2014;124(19):3007--15.

10. Bubolz AM, Weissinger SE, Stenzinger A, Arndt A, Steinestel K,
Brüderlein S, et al. Potential clinical implications of BRAF mutations
in histiocytic proliferations. Oncotarget. 30 de junho de
2014;5(12):4060--70.

11. Nelson DS, Quispel W, Badalian-Very G, van Halteren AGS, van den Bos
C, Bovée JVMG, et al. Somatic activating ARAF mutations in Langerhans
cell histiocytosis. Blood. 15 de maio de 2014;123(20):3152--5.

12. Jang S, Atkins MB. Treatment of BRAF-mutant melanoma: the role of
vemurafenib and other therapies. Clin Pharmacol Ther. janeiro de
2014;95(1):24--31.

13. Donadieu J, Larabi IA, Tardieu M, Visser J, Hutter C, Sieni E, et
al. Vemurafenib for Refractory Multisystem Langerhans Cell Histiocytosis
in Children: An International Observational Study. J Clin Oncol.
1\textsuperscript{o} de novembro de 2019;37(31):2857--65.

14. Evseev D, Kalinina I, Raykina E, Osipova D, Abashidze Z, Ignatova A,
et al. Vemurafenib provides a rapid and robust clinical response in
pediatric Langerhans cell histiocytosis with the BRAF V600E mutation but
does not eliminate low-level minimal residual disease per ddPCR using
cell-free circulating DNA. Int J Hematol. dezembro de
2021;114(6):725--34.

15. Badalian-Very G, Vergilio JA, Degar BA, MacConaill LE, Brandner B,
Calicchio ML, et al. Recurrent BRAF mutations in Langerhans cell
histiocytosis. Blood. 16 de setembro de 2010;116(11):1919--23.

16. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al.
Mutations of the BRAF gene in human cancer. Nature. 27 de junho de
2002;417(6892):949--54.

17. Brown NA, Furtado LV, Betz BL, Kiel MJ, Weigelin HC, Lim MS, et al.
High prevalence of somatic MAP2K1 mutations in BRAF V600E-negative
Langerhans cell histiocytosis. Blood. 4 de setembro de
2014;124(10):1655--8.

18. Nelson DS, van Halteren A, Quispel WT, van den Bos C, Bovée JVMG,
Patel B, et al. MAP2K1 and MAP3K1 mutations in Langerhans cell
histiocytosis. Genes Chromosomes Cancer. junho de 2015;54(6):361--8.

19. Hutter C, Minkov M. Insights into the pathogenesis of Langerhans
cell histiocytosis: the development of targeted therapies. ImmunoTargets
Ther. 2016;5:81--91.

20. Héritier S, Jehanne M, Leverger G, Emile JF, Alvarez JC, Haroche J,
et al. Vemurafenib Use in an Infant for High-Risk Langerhans Cell
Histiocytosis. JAMA Oncol. setembro de 2015;1(6):836--8.

21. Gerbaux M, Diallo S, Dedeken L, Dangoisse C, Bott A, Heritier S, et
al. Effective rescue treatment with vemurafenib of an infant with
high-risk Langerhans cell histiocytosis. Ann Dermatol Vénéréologie.
novembro de 2020;147(11):782--5.

22. Donadieu J, Bernard F, van Noesel M, Barkaoui M, Bardet O, Mura R,
et al. Cladribine and cytarabine in refractory multisystem Langerhans
cell histiocytosis: results of an international phase 2 study. Blood. 17
de setembro de 2015;126(12):1415--23.

23. Kudo K, Maeda M, Suzuki N, Kanegane H, Ohga S, Ishii E, et al.
Hematopoietic Stem Cell Transplantation in Children with Refractory
Langerhans Cell Histiocytosis. Blood. 29 de novembro de
2018;132(Supplement 1):4657--4657.

24. Cohen Aubart F, Emile JF, Carrat F, Charlotte F, Benameur N,
Donadieu J, et al. Targeted therapies in 54 patients with
Erdheim-Chester disease, including follow-up after interruption (the
LOVE study). Blood. 14 de setembro de 2017;130(11):1377--80.

25. Tardieu M, Néron A, Duvert-Lehembre S, Amine Larabi I, Barkaoui M,
Emile JF, et al. Cutaneous adverse events in children treated with
vemurafenib for refractory BRAF(V600E) mutated Langerhans cell
histiocytosis. Pediatr Blood Cancer. setembro de 2021;68(9):e29140.

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