Molecular and clinicopathologic characterization of
pediatric histiocytoses

Zofia Hélias-Rodzewicz, Jean Donadieu, Nathalie Terrones, Mohamed Aziz Barkaoui, Anne Lambilliotte, Despina Moshous, Caroline J.C. Thomas, Saba Azarnoush, Marlène Pasquet, Ludovic Mansuy, et al.

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To cite this version:

Zofia Hélias-Rodzewicz, Jean Donadieu, Nathalie Terrones, Mohamed Aziz Barkaoui, Anne Lambilliotte, et al. Molecular and clinicopathologic characterization of pediatric histiocytoses. American Journal of Hematology, 2023, 98 (7), pp.1058-1069. 10.1002/ajh.26938. hal-04191726

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# Molecular and clinicopathologic characterization of pediatric histiocytoses

Zofia Hélias-Rodzewicz¹

Jean Donadieu²

Nathalie Terrones¹

Mohamed-Aziz Barkaoui²

Anne Lambilliotte³

Despina Moshous⁴

Caroline Thomas⁵

Saba Azarnous⁶

Marlène Pasquet⁷

Ludovic Mansuy⁸

Nathalie Aladjidi⁹

Eric Jeziorski¹⁰

Perrine Marec-Berard¹¹

Marion Gilibert-Yvert¹²

Alexandra Spiegel¹³

Paul Saultier¹⁴

Isabelle Pellier¹⁵

Anne Pagnier¹⁶

Sophie Pertuisel¹⁷

Maryline Poiree¹⁸

Damien Bodet¹⁹

Frédéric Millot²⁰

Florentina Isfan²¹

Jean-Louis Stephan²²

Amaury Leruste²³

Charlotte Rigaud²⁴

Bruno Filhon²⁵

Liana Carausu²⁶

Yves Reguerre²⁷

Isabelle Kieffer²⁸

Bénédicte Brichard²⁹

Rim Ben Jannet¹

Mariama Bakari¹

Ahmed Idbaih³⁰

Christine Bodemer³¹

Fleur Cohen-Aubart³²

Julien Haroche³²

Abdellatif Tazi³³

Sabah Boudjemaa³⁴

Sylvie Fraitag³⁵

Jean-François Emile¹

<figure><img src="image_3.png" /></figure>

Sébastien Heritier²

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for the French Histiocytosis Study Group

## Correspondence

Sébastien Heritier, French Reference Center for Histiocytosis, Department of Pediatric Hematology and Oncology, Trousseau Hospital, AP-HP, Sorbonne Université, Paris, France.

Email: sebastien.heritier@aphp.fr

## Funding information

Conquer Cancer Foundation ASCO Young Investigator Award funded by the Strike 3 Foundation, Grant/Award Number: 11413; Programme de Recherche Translationnelle en Cancérologie, Grant/Award Number: 19-143

## Abstract

The spectrum of somatic mutations in pediatric histiocytoses and their clinical implications are not fully characterized, especially for non-Langerhans cell histiocytosis (-LCH) subtypes. A cohort of 415 children with histiocytosis from the French histiocytosis registry was reviewed and analyzed for *BRAF*<sup>V600E</sup>. Most *BRAF*<sup>WT</sup> samples were analyzed by next-generation sequencing (NGS) with a custom panel of genes for histiocytosis and myeloid neoplasia. Of 415 case samples, there were 366 LCH, 1 Erdheim-Chester disease, 21 Rosai-Dorfman disease (RDD), 21 juvenile xanthogranuloma (JXG, mostly with severe presentation), and 6 malignant histiocytosis (MH). *BRAF*<sup>V600E</sup> was the most common mutation found in LCH (50.3%, *n* = 184). Among 105 non-*BRAF*<sup>V600E</sup>-mutated LCH case samples, NGS revealed mutations as follows: MAP2K1 (*n* = 44), *BRAF* exon 12 deletions (*n* = 26), and duplications (*n* = 8), other *BRAF* V600 codon mutation (*n* = 4), and non-MAP-kinase pathway genes (*n* = 5). Wild-type sequences were identified in 17.1% of samples. *BRAF*<sup>V600E</sup> was the only variant significantly correlated with critical presentations: organ-risk involvement and

Jean-François Emile and Sébastien Heritier contributed equally to this study.

For affiliations refer to page 1067

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
© 2023 The Authors. *American Journal of Hematology* published by Wiley Periodicals LLC.
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neurodegeneration. MAP-kinase pathway mutations were identified in seven RDD (mostly MAP2K1) and three JXG samples, but most samples were wild-type on NGS. Finally, two MH samples had KRAS mutations, and one had a novel BRAF<sup>G469R</sup> mutation. Rarely, we identified mutations unrelated to MAP-kinase pathway genes. In conclusion, we characterized the mutational spectrum of childhood LCH and clinical correlations of variants and subtypes. Variants responsible for JXG and RDD were not elucidated in more than half of the cases, calling for other sequencing approaches.

# 1 | INTRODUCTION

Histiocytosis constitutes a group of rare disorders arising from tissue infiltration by myeloid cells with diverse macrophage or dendritic cell phenotypes.¹ Oncogenic somatic mutations of the MAP-kinase pathway have been reported in varying frequencies depending on histiocytosis subgroup, age of the studied population, and sensitivity of the molecular method used.

Langerhans cell histiocytosis (LCH) and Erdheim-Chester disease (ECD) are included among the L group histiocytoses in the 2016 revised classification² because of their shared clinical and molecular characteristics. LCH is by far the most common histiocytosis with a pediatric predominance, whereas ECD is very rare in children. For this L group, the mutational spectrum is the best described, and molecular alterations involving the MAP-kinase pathway are assumed to be identifiable in about three of every four cases. In childhood LCH, BRAF<sup>V600E</sup> is the most frequently identified mutation (about 50%), followed by alterations in exons 2 and 3 of MAP2K1, which are found in ~11%-30% of pediatric cases.³⁻⁸ Recently, small deletions and duplications of BRAF exon 12 have been reported, but their prevalence is less clearly established.⁹,¹⁰ Data are even more scarce for the mutational spectrum of less frequent histiocytoses such as juvenile xanthogranuloma (JXG), Rosai-Dorfman disease (RDD), and malignant histiocytosis (MH).⁷,¹¹,¹²

The mutation status related to the MAP-kinase pathway is of therapeutic interest because of implications for which targeted therapy is indicated for chemotherapy-resistant disease: specific inhibitors of BRAF in the context of the BRAF<sup>V600E</sup> mutation and MEK inhibitors for other BRAF or MAP2K1 mutations.¹ Moreover, we have established a genotype-phenotype correlation in childhood LCH between the BRAF<sup>V600E</sup> mutation and multisystem (MS) risk organs positive (RO+) LCH, resistance to first-line vinblastine-steroid chemotherapy, reactivations, and late sequelae, in particular neurodegenerative ramifications.¹³,¹⁴ For pediatric LCH, these data have paved the way to developing tailored strategies of therapeutic stratification and long-term adapted follow-up based on molecular findings.

Molecular analysis in histiocytoses represents a difficulty compared with other neoplasia pathologies because of the low infiltration into lesions of pathological cells bearing the oncogenic mutation and the limited material from diagnostic biopsies in young children.⁴,¹⁵ Centralized analysis in a reference pathology laboratory is of interest

in order to benefit from an optimized process that includes histological review, macrodissection if necessary to enrich the sample in pathological histiocytosis, and finally the choice of analytical method, with digital PCR if necessary.¹³

This study used data from a representative cohort of children treated for pediatric histiocytosis, and whose molecular analysis was centralized in a national expert reference laboratory. The aim was to define the spectrum of somatic mutations for each histiocytosis subtype and characterize genotype-phenotype correlations, with a specific focus on non-BRAF<sup>V600E</sup> mutations in childhood LCH and on mutations associated with JXG, RDD, and MH (i.e., non-LCH subtypes).

# 2 | MATERIALS AND METHODS

## 2.1 | Patients

Among patients with pediatric histiocytosis included in the French National histiocytoses registry,¹⁶ 484 had benefited from the histologic review over the last 12 years (2010–2021) in the histiocytosis French reference laboratory. The inclusion criteria were age <18 years at diagnosis, presence of histiocytes in analyzed material, and amplifiable DNA detected after extraction. The exclusion criteria were the absence of histiocytosis infiltration in samples or non-amplifiable DNA. This study was approved by the Ethics Committee, Sud-Ouest et Outre-Mer II (#2019-A01814-53), and was conducted in accordance with the Declaration of Helsinki. All patients provided written informed consent (clinical trial registration NCT04437381 [Molecular Targets for the Treatment of Histiocytosis TARGET-HISTIO]). Among the 484 pediatric biopsy samples received during the covered period, 415 were from cases that met the inclusion criteria (Figure 1).

Demographic data, clinical characteristics, extent of disease, type of treatment and response to therapy, reactivation, and permanent sequelae were recorded as previously described¹³ and according to classifications established by the Histiocyte Society.¹⁷ Treatment efficacy was evaluated according to the classification used by the Histiocyte Society. When tumorous lesions were present, we used the Response Evaluation Criteria in Solid Tumors version 1.1, as previously described.¹⁸ Among childhood, LCH patients included here, 214/366 (58.5%, Supplementary Material 1A) were already included
**484 biopsies from children with histiocytosis selected for central review and DNA sequencing in a French expert reference center (years, 2010-2021)**

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**415 samples with confirmed diagnosis and studied for BRAF<sup>V600E</sup>**

- LCH n = 366

- ECD n = 1

- RDD n = 21

- JXG n = 21

- MH n = 6

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**148 samples analyzed by next-generation sequencing (NGS)**

with a panel of genes customized for histiocytosis and myeloid neoplasia

**FIGURE 1** Workflow of recruitment of histiocytosis patients into the study. ECD, Erdheim-Chester disease; JXG, juvenile xanthogranuloma; LCH, Langerhans cell histiocytosis; MH, malignant histiocytosis; NGS, next-generation sequencing; RDD, Rosai-Dorfman disease.

in the *BRAF*<sup>V600E</sup> correlation study published in 2016 by our group, with extended follow-up.<sup>13</sup>

## 2.2 | Histological review and DNA extraction

Histologic control with hematoxylin and eosin staining and CD1a (Dako, clone 010, #IR069), CD207 (Leica, #NCL-L-Langerin), and CD163 (Leica, clone 10D6, #NCL-CD163) antibody analysis was performed to review the histiocytic disorder diagnosis and identify a region suitable for DNA extraction. Tissue samples were macrodissected and the percentage of histiocytes was evaluated for each patient. Since 2016, pERK (Cell Signaling, CD13.14.4E, XP®, #8544) antibody has progressively been applied to complete immunohistochemistry (IHC) analysis.

For most samples, DNA was extracted as described by Colomba et al.<sup>19</sup> Since 2020, DNA extraction has been automated on a Maxwell® RSC Instrument (Promega, France), with extraction performed according to the supplier's recommendations. For formalin-fixed, paraffin-embedded (FFPE) and frozen biological materials, the Maxwell® RSC DNA FFPE Kit and Maxwell® RSC Tissue DNA Kit were used, respectively. For targeted *BRAF*<sup>V600</sup> analysis, DNA was quantified by a spectrophotometric method (ND-100, Nanodrop®, or Multi-scan Go, Thermo Fisher Scientific, France). For next-generation sequencing (NGS) analysis, DNA quantity was evaluated by the Qubit Fluorometer (Thermo Fisher Scientific, France).

## 2.3 | *BRAF*<sup>V600</sup> analysis

*BRAF*<sup>V600</sup> status was defined using first-line detection methods (qPCR or pyrosequencing), as previously described,<sup>13,19</sup> and reviewed using a digital PCR approach if the first result was doubtful.<sup>20</sup> Since 2021, we

have applied a new digital PCR method using a QIAcuity One, 5plex Instrument (Qiagen) with a commercially available *BRAF*<sup>V600E</sup> assay (#DMH0000004, Qiagen). The reaction conditions for this digital PCR are reported in Supplementary Material 1B.

## 2.4 | DNA-Seq NGS

The DNA-Seq panel included almost 60 genes covering hot spots or all exons previously reported to be mutated in histiocytoses and genes involved in the MAP-kinase pathway and myeloid neoplasia. The sequencing technique used and the regions covered by panels used in this study are presented in Supplementary Material 1C-E. The quality sequencing data and target coverage quality data are also included. The sequencing data were analyzed depending on the applied technique. Mutations detected by DNA sequencing were interpreted according to standards and guidelines as described by Richards et al.,<sup>21</sup> and Sanger sequencing and length analysis of PCR products (LAPP) methods were used to confirm unusual and/or low-frequency *BRAF* and *MA2K1* alterations, as described in Supplementary Material 1F,G.

## 2.5 | Statistical analysis

Differences between groups were tested with the Mann-Whitney U test for quantitative variables and Fisher's exact test for qualitative variables. For statistical analysis, the threshold significance was 0.01, and for univariate analyses (multitests) of LCH presentation according to mutational status, *p* < .002 was considered statistically significant (Bonferroni correction). Survival analyses included the interval between diagnosis and an event (reactivation or death) or the last examination. Survival rates were estimated with the Kaplan-Meier
<figure><img src="image_8.png" /><figcaption>**FIGURE 2** Molecular overview of pediatric histiocytoses. (A) Frequency of *BRAF*<sup>V600E</sup> mutation in different histiocytosis disorders. The sole ECD *BRAF*<sup>V600E</sup> patient is not represented. (B) Landscape and frequency of different mutations observed by DNA-Seq in histiocytosis cases that were wild-type (WT) for *BRAF*<sup>V600E</sup>. The section results are given in %. DUP, duplication; INS, insertion; PM, point mutation. [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

method, and subgroups were compared with the log-rank test. All sta-
tistical analyses were performed with Stata 13 software. The cut-off
date for these analyses was June 31, 2022.

almost all patients analyzed by NGS for MH had at least one non-
recurrent molecular alteration (Figure 2B). A detailed description of all
detected mutations is presented in Supplementary Table 1.

3 | RESULTS

A total of 415 biopsies were analyzed from 415 children with confirmed diagnoses as follows: LCH, 366 patients; ECD, 1 patient; RDD, 21 patients; JXG, 21 patients; and MH, 6 patients. The *BRAF*<sup>V600E</sup> mutation was detected in 45.1% of 415 samples (n = 187/415) and almost exclusively in cases of LCH. Three non-LCH histiocytosis samples showed *BRAF*<sup>V600E</sup> mutations, two from RDD and the ECD case (Figure 2A).

Because of DNA quantity and quality, DNA-Seq analysis was not
performed in 80 BRAF<sup>WT</sup> (wild-type) samples. Among 148 samples
that were wild-type at the <i>BRAF</i><sup>V600E</sup> locus and analyzed by DNA-
Seq, 26.4% still had other aberrations in <i>BRAF</i> (n = 39/148), mostly
deletions and small duplications localized in exon 12, and 33.1% had
mutations in the <i>MAP2K1</i> gene (n = 49/148). Among <i>MAP2K1</i> alter-
ations, exon 2 deletions were more frequent than exon 3 deletions
(57.2% vs. 30.6% of all <i>MAP2K1</i> alterations), whereas point mutations
were rare although seen in both exons (6.1% in exon 2 and 6.1% in
exon 3, of all <i>MAP2K1</i> alterations). In 9.5% of samples (n = 14/148),
we detected a mutation in genes other than <i>BRAF</i> and <i>MAP2K1</i>, and
in three cases, these mutations coexisted with <i>BRAF</i> or <i>MAP2K1</i vari-
ants. For both RDD and JXG samples, <i>MAP2K1</i> and <i>KRAS</i> mutations
were occasionally found, but for most of these histiocytosis subtypes,
no mutation was identified despite our large NGS panel. Finally,

3.1 | Molecular status among children with LCH and clinical correlation

Among 366 children with LCH investigated for *BRAF*<sup>V600E</sup>, 184 (50.3%) harbored this mutation. Among those with non-*BRAF*<sup>V600E</sup>-mutated LCH (n = 182), 105 samples were subjected to further molecular analysis. In this subgroup, the prevalence of other somatic mutations was 41.9% for *MAP2K1* exon 2 or 3 mutations (n = 44), 24.8% for a small deletion in *BRAF* exon 12 (n = 26), and 7.6% for a small duplication in *BRAF* exon 12 (n = 8) (Figure 2B, Supplementary Table 1). Four patients had a *BRAF* mutation localized at the V600 codon that was different from V600E (V600_W604delinsKTDG, V600_K601delinsEISMEWVP, V600delinsDL, and V600D). Four other patients had non-recurrent mutations without other molecular alterations: CSF1R<sup>Q965+</sup> (variant allele frequency [VAF], 3.4%) together with EZH2<sup>P7515</sup> (VAF, 15.0%), ETV6<sup>R369W</sup> (VAF, 8.2%), NF1<sup>R1250W</sup> (VAF, 4.2%), and TET2<sup>Q810*</sup> (VAF, 12.2%). In two children, MAP2K1 deletion was found together with either JAK2<sup>R677R</sup> (VAF, 10.2%) or GATA2<sup>G135_P137del</sup> (VAF, 10.0%) mutation. Thus, we sometimes but rarely observed alterations in genes commonly mutated in myeloid malignancies or clonal hematopoiesis, in contrast to what is reported in adults with ECD.<sup>22</sup>

Finally, no significant mutation was identified after NGS analysis
in 19/105 samples (18.1% of non-*BRAF*<sup>V600E</sup>-mutated samples).
*BRAF*<sup>V600E</sup> mutation was associated with multisystem (MS) disease
<table><caption>TABLE 1 Characteristics of LCH patients according to BRAF<sup>V600E</sup> status; values in % of *n* unless otherwise indicated.</caption><thead><tr><th>Characteristics</th><th>With BRAF<sup>V600E</sup> (n = 184)</th><th>Without BRAF<sup>V600E</sup> (n = 182)</th><th>p</th></tr></thead><tbody><tr><td>Male</td><td>51.6</td><td>64.8</td><td>.01</td></tr><tr><td>Female</td><td>48.4</td><td>35.2</td><td></td></tr><tr><td>Age at diagnosis (median)</td><td>1.9 years</td><td>3.3 years</td><td>.002</td></tr><tr><td>SS-LCH</td><td>51.1</td><td>72.5</td><td>&lt;.001</td></tr><tr><td>RO- MS LCH</td><td>21.7</td><td>19.8</td><td></td></tr><tr><td>RO+ MS LCH</td><td>27.2</td><td>7.7</td><td></td></tr><tr><td>Bone involvement</td><td>72.3</td><td>73.6</td><td>.81</td></tr><tr><td>Skin involvement</td><td>56.0</td><td>30.2</td><td>&lt;.001</td></tr><tr><td>Pituitary involvement</td><td>19.6</td><td>11.5</td><td>.04</td></tr><tr><td>Tumor of central nervous system involvement</td><td>5.4</td><td>3.3</td><td>.44</td></tr><tr><td>Liver involvement</td><td>21.7</td><td>4.9</td><td>&lt;.001</td></tr><tr><td>Hematologic involvement</td><td>22.3</td><td>3.8</td><td>&lt;.001</td></tr><tr><td>Spleen involvement</td><td>19.6</td><td>4.4</td><td>&lt;.001</td></tr><tr><td>Lung involvement</td><td>12.5</td><td>12.1</td><td>1</td></tr><tr><td>Lymph node involvement</td><td>10.3</td><td>11.0</td><td>.87</td></tr><tr><td>Follow-up (median)</td><td>5.1 years</td><td>3.3 years</td><td>.008</td></tr><tr><td>VLB steroid regimen</td><td>60.9</td><td>48.9</td><td>.03</td></tr><tr><td>Response to VLB steroid</td><td>76.8</td><td>94.4</td><td>.001</td></tr><tr><td>Receipt of second-line therapy</td><td>30.4</td><td>7.1</td><td>&lt;.001</td></tr><tr><td>Receipt of targeted therapy</td><td>18.5</td><td>3.3</td><td>&lt;.001</td></tr><tr><td>5-year cumulative incidence of reactivations</td><td>42.0</td><td>30.3</td><td>.06</td></tr><tr><td>Permanent consequence</td><td>25.5</td><td>15.9</td><td>.03</td></tr><tr><td>Neurodegeneration</td><td>7.6</td><td>0</td><td>&lt;.001</td></tr><tr><td>Sclerosing cholangitis</td><td>3.3</td><td>3.3</td><td>1</td></tr><tr><td>Severe lung involvement</td><td>1.6</td><td>2.7</td><td>.50</td></tr><tr><td>Death</td><td>2.7</td><td>1.6</td><td>.72</td></tr></tbody></table>

Abbreviations: LCH, Langerhans cell histiocytosis; MS, multiple system; RO, risk organs; SS, single system, VLB, vinblastine.

and RO involvement: 27.2% of patients with BRAF<sup>V600E</sup> had MS RO+ LCH compared with 7.7% of patients without BRAF<sup>V600E</sup> (*p* < .001), and hematologic, spleen, and liver involvement was present in 22.3%, 19.6%, and 21.7% of cases with BRAF<sup>V600E</sup> compared with 3.8%, 4.4%, and 4.9% of cases without this mutation (*p* < .001) (Table 1).

Patients with BRAF<sup>V600E</sup> LCH were more likely to have skin involvement (56.0%, BRAF<sup>V600E</sup>; 30.2%, non-BRAF<sup>V600E</sup>; *p* < .001) and to require more vinblastine-steroid chemotherapy (60.9%, BRAF<sup>V600E</sup>; 48.9%, non-BRAF<sup>V600E</sup>; *p* = .03). Response to vinblastine-steroid chemotherapy was lower with BRAF<sup>V600E</sup> (76.8%, BRAF<sup>V600E</sup>; 94.4%, non-BRAF<sup>V600E</sup>; *p* = .001), and 30.4% of those harboring BRAF<sup>V600E</sup> were treated with a second-line therapy compared with 7.1% without BRAF<sup>V600E</sup>. Only 3.3% (*n* = 6) of children without BRAF<sup>V600E</sup> received targeted therapy, compared with 18.5% (*n* = 34) bearing BRAF<sup>V600E</sup>. Response to targeted therapy was constant when administered for chemo-refractory active LCH disease. Patients with BRAF<sup>V600E</sup> tended to experience a higher 5-year LCH reactivation risk (42.0%) compared with those lacking BRAF<sup>V600E</sup> (30.3%; log-rank test, *p* = .06). Regarding long-term consequences of the disease, 7.6% of patients with

BRAF<sup>V600E</sup> developed a clinical neurodegenerative LCH, but no patients without this mutation did so (*p* < .001). For the less frequent recurrent mutations (MAP2K1, BRAF exon 12 small deletions, BRAF exon 12 small duplication), we found no significant difference related to characteristics of LCH disease and outcome between these subgroups (Table 2).

Although cases involving non-BRAF<sup>V600E</sup>-mutated LCH were globally less severe, some involved a life-threatening presentation. One patient with a BRAF exon 12 small duplication had an MS RO+ LCH (hematological involvement, disease activity score [DAS] = 6, diagnosis at age 0.3 years) but with a favorable outcome as the disease responded to vinblastine-corticosteroid therapy. Two patients with a BRAF exon 12 small deletion had a severe disease course, one with severe destructive lung involvement and the other with sclerosing cholangitis. Both underwent solid organ transplantation (lung and liver, respectively) with continuous MEK inhibitor therapy before, during, and after the procedure, with good graft function and no reactivation under targeted therapy. Three patients with a MAP2K1 mutation had a severe disease presentation. One of these children
<table><caption>TABLE 2 Characteristics of LCH samples that were wild-type for BRAF<sup>V600E</sup> and studied by NGS, according to molecular status; values in % of n unless otherwise specified.</caption><thead><tr><th>Characteristics</th><th>MAP2K1 mutation (n = 44)</th><th>BRAF exon 12 small deletion (n = 26)</th><th>BRAF exon 12 small duplication (n = 8)</th><th>WT (n = 19)</th><th>p</th></tr></thead><tbody><tr><td>Male</td><td>72.7</td><td>61.5</td><td>75.0</td><td>52.6</td><td>.40</td></tr><tr><td>Female</td><td>27.3</td><td>38.5</td><td>25.0</td><td>47.4</td><td></td></tr><tr><td>Age at diagnosis (median)</td><td>3.4 years</td><td>3.2 years</td><td>4.1 years</td><td>2.2 years</td><td>NS</td></tr><tr><td>SS-LCH</td><td>72.7</td><td>80.8</td><td>75.0</td><td>73.7</td><td></td></tr><tr><td>RO- MS LCH</td><td>25</td><td>11.5</td><td>12.5</td><td>21.0</td><td>.60</td></tr><tr><td>RO+ MS LCH</td><td>2.3</td><td>7.7</td><td>12.5</td><td>5.3</td><td></td></tr><tr><td>Bone involvement</td><td>88.6</td><td>61.5</td><td>100</td><td>63.2</td><td>.008</td></tr><tr><td>Skin involvement</td><td>18.2</td><td>30.8</td><td>12.5</td><td>47.4</td><td>.08</td></tr><tr><td>Pituitary involvement</td><td>9.1</td><td>11.5</td><td>0</td><td>15.8</td><td>.59</td></tr><tr><td>Tumor of the central nervous system involvement</td><td>0</td><td>3.8</td><td>0</td><td>5.3</td><td>.21</td></tr><tr><td>Liver involvement</td><td>2.3</td><td>7.7</td><td>0</td><td>0</td><td>.56</td></tr><tr><td>Hematologic involvement</td><td>2.3</td><td>0</td><td>12.5</td><td>5.3</td><td>.37</td></tr><tr><td>Spleen involvement</td><td>2.3</td><td>0</td><td>0</td><td>0</td><td>1.0</td></tr><tr><td>Lung involvement</td><td>9.1</td><td>15.4</td><td>12.5</td><td>10.5</td><td>.87</td></tr><tr><td>Lymph node involvement</td><td>22.7</td><td>3.8</td><td>12.5</td><td>5.3</td><td>.10</td></tr><tr><td>Follow-up (median)</td><td>1.6 years</td><td>2.6 years</td><td>2.2 years</td><td>1.6 years</td><td>NS</td></tr><tr><td>VLB steroid regimen</td><td>47.7</td><td>50.0</td><td>75.0</td><td>57.9</td><td>.54</td></tr><tr><td>Response to VLB steroid</td><td>95.2</td><td>91.7</td><td>80.0</td><td>90.9</td><td>.92</td></tr><tr><td>Receipt of second-line therapy</td><td>6.8</td><td>7.7</td><td>12.5</td><td>10.5</td><td>.81</td></tr><tr><td>Receipt of targeted therapy</td><td>4.5</td><td>7.7</td><td>0</td><td>5.2</td><td>.89</td></tr><tr><td>5-year cumulative incidence of reactivations</td><td>40.8</td><td>20.1</td><td>31.4</td><td>35.4</td><td>.60</td></tr><tr><td>Permanent consequence</td><td>11.4</td><td>19.2</td><td>0</td><td>15.8</td><td>.58</td></tr><tr><td>Neurodegeneration</td><td>0</td><td>0</td><td>0</td><td>0</td><td>-</td></tr><tr><td>Sclerosing cholangitis</td><td>0</td><td>7.7</td><td>0</td><td>0</td><td>.19</td></tr><tr><td>Severe lung involvement</td><td>4.5</td><td>7.7</td><td>0</td><td>0</td><td>.77</td></tr><tr><td>Death</td><td>0</td><td>0</td><td>0</td><td>5.3</td><td>.28</td></tr></tbody></table>

Note: When specified, p value was obtained by comparison among all four groups of patients.

Abbreviations: LCH, Langerhans cell histiocytosis; MS, multiple system; NS, not significant; RO, risk organs; SS, single system; WT, wild-type, VLB, vinblastine.

(MAP2K1<sup>D,F53C</sup>) had MS RO+ LCH (DAS = 13, diagnosis at age 0.4 years) with diffuse lung lesions refractory to vinblastine-corticosteroid therapy, but experienced a clinical complete remission with MEK inhibitor therapy. The second child with MAP2K1<sup>F53_Q58delinsL</sup> underwent a left complete pneumonectomy at age 1 month because of complete destruction of the unilateral lung parenchyma leading to serious mechanical complications, and the third of these patients with MAP2K1<sup>Q56_G61delinsR</sup> was successfully treated with MEK inhibitor after experiencing no response to vinblastine-corticosteroid first-line chemotherapy and severe diffuse lung lesions. The four patients treated with MEK inhibitors were still on therapy at the last visit, including one who experienced reactivation after discontinuation.

Among four patients with mutations localized at the V600 codon of BRAF other than V600E, one patient, with BRAF<sup>V600_W604delinsLTDG</sup>, had infant MS RO+ that responded to vinblastine-corticosteroid

therapy with a favorable outcome, while the three other patients had benign single-system (SS)-LCH disease.

Among patients with non-recurrent mutations, one with TET2<sup>Q810+</sup> and one with ETV6<sup>R369W</sup> had SS-LCH of skin and bone, respectively, with unremarkable presentation and outcome. Conversely, a patient with CSF1R<sup>Q965+</sup> and EZH2<sup>P751S</sup> combined mutations had SS bone LCH diagnosed at age 8.3 years but had an uncommon reactivation with ECD features (osteosclerosis of long bones, coated aorta) at age 14.5 years, managed with interferon-α therapy. A patient with NF1<sup>R1250W</sup> had MS RO+ LCH diagnosed at age 6.2 years, with a favorable outcome after vinblastine-corticosteroid treatment together with 6-mercaptopurine therapy. Finally, 19 patients had wild-type samples after DNA-targeted NGS investigation, and pERK IHC was positive in all cases. Of note, in this subgroup of patients, one had infant MS RO+ LCH refractory to vinblastine-steroid therapy but responsive to MEK inhibitor, and one
had indolent skin SS-LCH associated with severe inherited osteopo-
trosis, which ended in death.

3.2 | Molecular study of childhood RDD

Samples of 21 children with RDD were included in this study (Supplementary Table 2A), 10 boys and 11 girls with a median age of 7.9 years (range, 0.8-15.4 years) at diagnosis, 7 (33%) of whom had sub-Saharan African ancestry. Six patients (18.6%) had significantly associated disease: R6 and R9 had primary immunodeficiency, R3 and R15 had developmental delay, which was associated with a congenital heart defect for R15; R20 had Steinert myotonic dystrophy, and R21 had fragile X syndrome. Nine (43%) patients had classic nodal RDD, and 12 (57%) had extranodal involvement: seven in the head and neck (nasal cavity and paranasal sinuses mass, *n* = 5; thickened mucosa of larynx, *n* = 1; orbit, *n* = 1), two in the skin, two with central nervous system tumors, and one with multifocal bone involvement.

Molecular studies showed significant mutation in samples from eight patients (Figure 2, Supplementary Table 1). Patients R1 and R6 had classical nodal RDD mutated *BRAF*<sup>V600E</sup> and *KRAS*<sup>G13D</sup>, respectively. *BRAF*<sup>V600E</sup> was also detected in patient R21, who had spinal cord involvement. MAP2K1 was the most mutated gene reported in this cohort, with four mutations identified that represented 22% of cases subjected to NGS (D58_E62del, V60E, I103S, and C121S). The four RDD patients with MAP2K1 mutations (R3, R9, R15, and R18) had lymph node involvement, and patient R3 also had a paranasal sinus mass. Regarding clinical syndromic features of patient R15 and the allelic MAP2K1<sup>1103S</sup> frequency at 51%, a germline mutation was suspected and confirmed, and a diagnosis of cardiofaciocutaneous syndrome was made (de novo mutation). After targeted NGS, 61.1% of studied samples were found to be wild-type. Of note, the DNMT3A<sup>R749C</sup> mutation was identified in patient R9 with a unifocal skin lesion. IHC analyses of biopsy samples showed pERK positivity for all tested samples, including nine that were wild-type.

In this cohort, 12 patients had benefited from an initial course of
corticosteroid therapy, five of whom also had cytotoxic chemother-
apy. Of the 12, 83% (n = 10/12) experienced a response to this first-
line therapy, and five of them had reactivation after discontinuation.
Nine patients had benefited from second-line chemotherapy: 2CDA
(n = 3), MEK inhibitor (n = 2), BRAF inhibitor (n = 1), mercaptopurine
(n = 1), methotrexate-azathioprine (n = 1), interferon alpha (n = 1),
and rapamycin associated with rituximab in a patient with associated
primary immunodeficiency (n = 1). Responses were variable, but of
note, the three patients treated with targeted therapy also experi-
enced a response. Seven patients received no systemic therapy, with
favorable outcomes.

3.3 | Molecular study of JXG

The group of 21 children with JXG (9 boys and 12 girls) had mostly a
severe presentation with multifocal skin or extracutaneous

involvement (Supplementary Table 2B). Median age at diagnosis was
0.6 years (range, 0-11.8 years). Fifteen patients had skin involvement
(solitary skin lesions, n = 2; multifocal skin lesions, n = 13). Moreover,
15 patients had extra-cutaneous involvement. Four had orbital or
ophthalmologic localization (iris involvement, n = 1; infiltrative orbital
lesion, n = 3), and 11 had a tumor mass with various localizations:
liver (n = 5), soft tissue (n = 5), bone (n = 4, including two patients
with a compressive base skull tumor and two patients with multifocal
bone localizations), spleen (n = 3), lung (n = 2), mediastinal (n = 1),
and kidney (n = 1). Finally, five patients had hematologic involvement
with severe cytopenia (JX3, JX7, JX8, JX12, and JX20).

All cases were negative for *BRAF*<sup>V600E</sup> mutation. NGS showed sig-
nificant mutations in samples from four patients (Figure 2B, Supple-
mentary Table 1). Two different mutations of *KRAS* were identified:
*KRAS*<sup>Q70_Y71insWEYSAMRDQ</sup> in patient JX1 with multifocal skin involve-
ment; and *KRAS*<sup>G12R</sup> in patient JX12 with multifocal skin involvement
and an infiltrative orbital lesion. This child presented with Burkitt leu-
kemia at age 2.7 years (2 years after the multifocal skin JXG diagno-
sis), and the treatment of the leukemia resulted in effacement of
cutaneous lesions but did not prevent the development of infiltrative
orbital lesions. A MAP2K1<sup>Q58_E62del</sup> was identified in a newborn girl
(JX3) with a voluminous neck tumor and hematologic involvement.
Finally, a CSF1R<sup>G970+</sup> mutation was identified in a boy aged 11.8 years
 patient C4) with a solitary skin lesion, and the histologic diagnosis
was a C group histiocytosis without typical features of JXG. After tar-
geted NGS, 80% of the studied samples remained wild-type. IHC ana-
lyses of biopsy samples showed pERK positivity for 12 samples, and
4 were negative (C4, JX6, JX10, JX17).

Twelve patients had benefited from a first-line systemic chemo-
therapy, and among them, 11 were treated with an LCH-like regimen
with vinblastine associated with corticosteroid or mercaptopurine
therapy. An objective response was observed for six patients, and one
patient had stabilization of the disease. Four patients were treated
with a second-line therapy: three (JX1, JX9, and JX12) received MEK
inhibitors, with a frank clinical response for two and stabilization of
the disease for one; and JX7 experienced clinical remission and favor-
able outcome after 2CDA-aracytine second-line therapy in the con-
text of very severe systemic disseminated disease. Finally, one other
patient (JX20), also with very severe systemic disseminated disease,
was treated with 2CDA-aracytine as first-line therapy but died of
infectious complications in a situation with persistently active disease.
This patient was the only one among those with JXG who died.

3.4 | Malignant histiocytosis

Six children (three boys and three girls) with MH were included in this
study (Supplementary Table 2C). Median age at diagnosis was
12.4 years (range, 1.4-16.2 years). For three children, MH was diag-
nosed secondary to the management of B-cell acute lymphoblastic
leukemia (patients M1 and M5) and Burkitt leukemia (patient M3). At
the initial diagnosis, bones were involved in three patients (multifocal
lesions) treated for a previous lymphoid malignancy, while one patient
had disseminated central nervous system tumor lesions (M1), one had a soft tissue tumor with inguinal lymph node localization (M4), and one patient had a cervical lymph node localization (M6). For two patients (M4 and M5), no follow-up was available. The systemic chemotherapy scheme was variable for the four patients for whom follow-up was available. Three of them experienced a poor response to chemotherapy, with death at a median of 11 months from diagnosis (range, 9.6-24.9 months). M6 was the only survivor and experienced a response to topotecan-temozolomide (TOTEM) combined chemotherapy.

IHC for pERK was performed for three cases (M2, M5, and M6), and all were positive. All cases were negative for BRAF<sup>V600E</sup> mutations (Figure 2A). NGS was performed on samples from five cases, and mutations were highlighted in four (Figure 2B, Supplementary Table 1): a novel BRAF<sup>G469R</sup> mutation associated with NF1<sup>E1266*</sup> in a girl age 12.4 years with disseminated central nervous system tumor lesions (patient M2); KRAS<sup>Q61H</sup> in a boy age 12.8 years with MH of bone secondary to Burkitt leukemia (patient M3); ETV6<sup>M389I</sup> in a boy age 1.4 years with a soft tissue tumor lesion (patient M4); and KRAS<sup>A146P</sup> in a boy age 16.2 years with MH of bone secondary to B-cell acute lymphoblastic leukemia (patient M5). No PTPN11 alteration was detected in our patients, in contrast to what has been reported in adults with malignant histiocytoses,<sup>23</sup> whereas two of our three patients with previous B-cell lymphoid malignancies had KRAS mutations, as reported in adults with histiocytic sarcoma.<sup>24</sup>

## 4 | DISCUSSION

Here, we present the results of the molecular and clinicopathological characterization of 415 cases of pediatric histiocytosis, most of them (88%) of the LCH subtype. Analyses of clinical features, response to treatment, and prognosis were conducted in relation to the molecular profiles of the cases. To our knowledge, this group is the largest homogeneous cohort of children representing all histiocytosis subtypes published to date.

As in other cancers, the most frequent BRAF alteration that we found in our pediatric cohort was the BRAF<sup>V600E</sup> mutation. In the present study, BRAF<sup>V600E</sup> was found in 45.1% of all histiocytoses and in 50.3% of childhood LCH. This result is in accordance with previously published findings showing BRAF<sup>V600E</sup> in 19%-67.6% of pediatric LCH.<sup>3-6</sup> In four children with LCH in the current study, we observed more complex alterations (small deletion/duplication) around codon V600 (Supplementary Table 1). These mutations altered the physicochemical properties of this hydrophobic domain, leading to the activation of downstream effectors of the MAP-kinase pathway. Such complex rearrangements of the V600 region have rarely been described in histiocytosis<sup>25</sup> and are more frequent in other cancers.<sup>26,27</sup> The BRAF<sup>V600E</sup> mutation has rarely been detected in non-LCH pediatric histiocytosis.<sup>24,28-30</sup> In our series, we found this mutation in only two RDD patients and in one with ECD, which is a rare condition in childhood.<sup>31-33</sup> Deletions and duplications in exon 12 of BRAF were exclusively found in the LCH group and were present in 24.8%

(deletions) and 7.6% (duplications) of non-BRAF<sup>V600E</sup>-mutated cases. A pathogenic role of these deletions and duplications in MAP-kinase pathway activation has been confirmed,<sup>9,10</sup> and they can be targeted by BRAF or MEK inhibitors; however, the efficacy of MEK inhibitors needs further investigation in the context of MAP2K1 exon 3 deletions (also called class 3 mutants<sup>34</sup>) and BRAF exon 12 small duplications.<sup>10</sup> Finally, in a patient with MH, we report a novel BRAF<sup>G469R</sup> mutation of unknown pathogenic function; pERK staining was positive in this MH case.

In our cohort, MAP2K1 alterations were the most frequently detected mutations in non-BRAF<sup>V600E</sup> childhood LCH, occurring in 42% of these patients. In contrast to BRAF, where the V600E point mutation is predominant, deletions were more common in the MAP2K1 gene, especially in exon 2 (57.2% of all MAP2K1 alterations). Furthermore, we identified MAP2K1 alterations in 20% of RDD cases and in one patient with JXG. Previous publications have reported MAP2K1 mutations in 11%-30% of pediatric LCH patients<sup>5-7</sup> and more rarely in other histiocytosis types.<sup>8,12,35,36</sup> Because non-BRAF<sup>V600E</sup>-mutated LCH cases constitute half of our LCH cohort, we can estimate that MAP2K1 mutations are found in 21% of childhood LCH, in accordance with previously published data.

In 12 samples without BRAF or MAP2K1 alteration, NGS revealed a mutation in genes other than BRAF or MAP2K1 (Supplementary Table 1). Additionally, samples from two MAP2K1-mutated LCH cases and one BRAF<sup>G469R</sup>-mutated MH case harbored a second mutation in another gene. The “less common” mutated genes in our series were KRAS (n = 5), CSF1R, ETV6, and NF1 (each n = 2), and DNMT3A, EZH2, GATA2, JAK2, and TET2 (each n = 1), all of which could have pathological consequences.<sup>21</sup> According to previous studies, the following genes (already found in cancer cases) also are rarely found to be mutated in pediatric histiocytosis: ARAF,<sup>7,37,38</sup> CSF1R,<sup>7</sup> KRAS,<sup>11,39</sup> KIT,<sup>7,40</sup> MET,<sup>3,7</sup> NF1,<sup>7</sup> NRAS,<sup>7</sup> and TP53,<sup>3,11</sup> and quite rarely with MAP2K1 or BRAF alteration.<sup>3,35</sup>

KRAS mutations were found in JXG (n = 2), RDD (n = 1), and MH (n = 2) types. KRAS is a well-known oncogene, and regarding histiocytoses, KRAS mutations have been reported in secondary MH<sup>23,24,41</sup> and occasionally in RDD or JXG.<sup>11</sup> In contrast to the activating mutations in CSF1R reported by Durham et al.,<sup>7</sup> both CSF1R mutations detected in our series (LCH, n = 1; XGJ, n = 1) generate the stop codon in the c-CBL binding domain, which might lead to abnormal CSF1R ubiquitination and degradation by the interruption of the c-CBL-CSF1R association.<sup>42,43</sup> The pathological mechanism of these mutants needs further investigation.

In contrast to reports concerning adults with ECD,<sup>22</sup> we observed very rare alteration in genes commonly mutated in myeloid malignancies or clonal hematopoiesis: the DNMT3AR<sup>749C</sup> mutation highlighted here in a child with RDD and the TET2<sup>Q810*</sup> mutation identified in a child with LCH were the only ones found in this cohort that are involved in clonal hematopoiesis.

Together with genetic analysis, we evaluated the correlation between the molecular profile and the clinical characteristics and outcomes for each histiocytosis subtype. Given the small number of cases representing the non-LCH histiocytosis subtypes and no recurrent
molecular profile found among them, a correlation analysis could not be performed in these groups. Nevertheless, we describe a substantial cohort of children with RDD, JXG, or MH with detailed clinical phenotype and outcomes, whereas most relevant previous publications have been case reports.

We note that the 21 children with JXG reported here typically had a severe presentation with multifocal skin or extracutaneous involvement. Only two patients had a solitary skin lesion, which is the most common presentation of JXG, with a favorable prognosis and spontaneous regression. To date, the largest study included 55 JXG patients,⁷ most with skin involvement only (n = 51), and with various molecular alterations reported but no clear predominance of any of them. The most common were changes in MAP2K1 (n = 6), CSF1R (n = 5), and KRAS (n = 4), and NTRK1 (n = 6) and BRAF (n = 4) gene fusions. In the current work, we also found associations with molecular changes in MAP2K1, CSF1R, and KRAS. Of interest, we identified no BRAF<sup>V600E</sup> mutations, in keeping with the findings of Durham et al., although this mutant has previously been reported in the central nervous system JXG.²⁹,³⁰ Further detailed clinico-molecular studies are needed to gain more insights into JXG. Furthermore, we report here one of the largest cohorts of childhood LCH cases with molecular analysis data. In this current study, we again show the clinical correlation of BRAF<sup>V600E</sup> mutation with MS RO+ LCH and resistance to first-line vinblastine-steroid chemotherapy that we reported in 2016,¹³ now with a patient cohort enriched by more than one-third with new patients, and a centralized molecular analysis, in contrast to our previous study. The risk of permanent sequelae, in particular clinical neurodegenerative LCH, is also significantly higher in BRAF<sup>V600E</sup>-mutated cases.¹⁴ Others have reported similar findings for pediatric LCH, in part, but in smaller cohorts.⁴,³⁷,⁴⁴ One exception is a recently published international cohort study by Kemps et al., which included an equivalent number of patients to ours.⁵ Regarding these data for children diagnosed with BRAF<sup>V600E</sup>-mutated LCH, specific attention should be paid to young children regarding MS-RO+ LCH risk. We suggest a close follow-up at least during the first year after the diagnosis, and parents should be made aware of symptoms that are suggestive of disease progression towards MS OR+ LCH (prolonged fever and/or diarrhea, signs of cytopenia, enlarged abdomen), with instructions to consult quickly if symptoms arise. Also, for children with BRAF<sup>V600E</sup>-mutated LCH, especially those who have experienced pituitary, skull base, or orbit bone involvement, follow-up after remission of LCH should be extended, if possible, throughout the primary and secondary school years to identify suggestive early signs of neurodegeneration (academic delay, attention, and concentration difficulties).

Despite the large cohort in the current study, groups with MAP2K1 mutations or BRAF exon 12 small deletions or duplications encompassed fewer than 50 patients each, which is a limitation for correlation analysis between these molecular subgroups. These cases are generally less severe than those involving BRAF<sup>V600E</sup> but without important differences between them. A few patients may have rare but life-threatening presentations, such as MS RO+ or severe lung involvement, or sclerosing cholangitis. In patients with MAP2K1

mutations (non-class 3 MAP2K1 mutants) or a BRAF exon 12 small deletions, MEK inhibitors were occasionally used with good clinical response, as previously reported with BRAF inhibitors for chemo-refractory BRAF<sup>V600E</sup>-mutated LCH.⁴⁵ Despite this broad molecular study, no mutation was detected in many cases. We found estimated wild-type rates of 9% for LCH, 60% for RDD, 80% for JXG, and 20% for MH, based on targeted NGS results. It should be pointed out that for LCH and RDD, activation of the MAPK pathway appears to be universal, as attested by ERK phosphorylation in all samples studied here. Unidentified genetic alterations or extracellular signals may be involved in wild-type samples. Further molecular analyses are needed (e.g., whole genome sequencing or fusion gene analyses performed on biopsy samples, or peripheral blood mononuclear cell NGS analyses for cases with very low infiltration of pathological histiocytes) to elucidate the genetic basis of this disease and improve genotype-phenotype correlations. As an example, a few publications have reported the presence of tyrosine kinase receptor fusion genes⁷,⁴⁶ or BRAF fusion⁹ in some pediatric histiocytosis, which is an avenue worth pursuing. Also, because of the rarity of pediatric histiocytosis or disseminated JXG, international teamwork probably will be necessary to accelerate progress in understanding this histiocytosis subtype.

## 5 | CONCLUSIONS

Our molecular analysis revealed a very heterogeneous mutational profile across histiocytosis subtypes. The genotype-phenotype correlation analysis confirmed our previous observations and showed a significant correlation of BRAF<sup>V600E</sup> mutation with MS RO+ LCH and neurodegeneration. Correlation analyses of non-BRAF<sup>V600E</sup>-mutated LCH cases harboring other genetic alterations in BRAF and MAP2K1 genes were harder to perform because of the small number of cases in each mutation subgroup; however, the prognosis appears to be better in these cases. Molecular and clinical information about these diseases offers important knowledge for clinicians to better evaluate disease prognosis and use in clinical management and therapeutic decision-making. Collaboration between groups of the Histiocytosis Society and among members of the European Consortium for Histiocytosis or North American Consortium for Histiocytosis may accelerate the application of the molecular knowledge to clinical practice and validate its usefulness.

## AUTHOR CONTRIBUTIONS

**Conception and design:** Jean Donadieu, Jean-François Emile, Sébastien Heritier, Zofia Hélias-Rodzewicz. **Development of methodology:** Jean-François Emile, Zofia Hélias-Rodzewicz. **Acquisition of data (Collection and examination of patient data):** Mohamed-Aziz Barkaoui, Anne Lambilliotte, Despina Moshous, Caroline Thomas, Saba Azarnoush, Marlène Pasquet, Ludovic Mansuy, Nathalie Aladjidi, Eric Jeziorski, Perrine Marec-Berard, Marion Gilibert-Yvert, Alexandra Spiegel, Paul Saultier, Isabelle Pellier, Anne Pagnier, Sophie Pertuisel, Maryline Poirée, Damien Bodet, Frédéric Millot, Florentina Isfan, Jean-Louis Stephan, Amaury Leruste, Charlotte Rigaud, Bruno Filhon, Liana
<figure><img src="image_9.png" /></figure>

Carausu, Yves Reguerre, Isabelle Kieffer, Bénédicte Brichard, Ahmed Idbaih, Christine Bodemer, Fleur Cohen-Aubart, Julien Haroche, Abdellatif Tazi, Sabah Boudjemaa, Sylvie Fraitag, Jean Donadieu, Jean-François Emile, Sébastien Heritier. *Experiments*: Nathalie Terrones, Rim Ben Jannet, Mariama Bakari, Zofia Hélias-Rodzewicz. *Analysis and interpretation of data* (e.g., statistical analysis, sequencing data analysis): Jean-François Emile, Zofia Hélias-Rodzewicz. *Writing, review, and/or revision of the manuscript*: Sébastien Heritier, Zofia Hélias-Rodzewicz. All authors approved the final version of the manuscript.

## AFFILIATIONS

¹EA4340 BECCOH, Pathology Department, Ambroise Paré Hospital, AP-HP, Université Paris-Saclay, Boulogne-Billancourt, France

²French Reference Center for Histiocytosis, Department of Pediatric Hematology and Oncology, Trousseau Hospital, AP-HP, Sorbonne Université, Paris, France

³Department of Pediatric Hematology and Oncology, Centre Hospitalo-Universitaire de Lille, Lille, France

⁴Department of Pediatric Immunology, Hematology and Rheumatology, Necker Hospital, AP-HP, Centre-Université Paris Cité, Institut Imagine, Paris, France

⁵Department of Pediatric Hematology and Oncology, Centre Hospitalo-Universitaire de Nantes, Nantes, France

⁶Department of Pediatric Immunology and Hematology, Robert Debré Hospital, AP-HP, Nord-Université Paris Cité, Paris, France

⁷Department of Pediatric Hematology and Oncology, Centre Hospitalo-Universitaire de Toulouse, Toulouse, France

⁸Department of Pediatric Hematology and Oncology, Centre Hospitalo-Universitaire de Nancy, Vandœuvre-lès-Nancy, France

⁹Department of Pediatric Hematology and Oncology, Centre d'Investigation Clinique (CIC) 1401, INSERM, Centre Hospitalo-Universitaire de Bordeaux, Bordeaux, France

¹⁰Department of Paediatric, Hôpital Arnaud de Villeneuve, Centre Hospitalo-Universitaire de Montpellier, Montpellier, France

¹¹Department of Paediatric Oncology, Institut d'Hémato-Oncologie Pediatrique, Lyon, France

¹²Department of Pediatric Hematology and Oncology, Centre Hospitalo-Universitaire de Tours, Tours, France

¹³Department of Pediatric Hematology and Oncology, Centre Hospitalo-Universitaire de Strasbourg, Strasbourg, France

¹⁴Department of Pediatric Hematology, Immunology and Oncology, Aix Marseille, APHM University, INSERM, INRAe, C2VN, La Timone Children's Hospital, Marseille, France

¹⁵Department of Pediatric Hematology and Oncology, Centre Hospitalo-Universitaire de Angers, Angers, France

¹⁶Department of Pediatric Hematology and Oncology, Centre Hospitalo-Universitaire de Grenoble, Grenoble, France

¹⁷Department of Pediatric Hematology and Oncology, Centre Hospitalo-Universitaire de Rennes, Rennes, France

¹⁸Department of Pediatric Hematology and Oncology, Centre Hospitalo-Universitaire de Nice, Nice, France

¹⁹Department of Pediatric Hematology and Oncology, Centre Hospitalo-Universitaire de Cean, Cean, France

²⁰Department of Pediatric Hematology and Oncology, Centre Hospitalo-Universitaire de Poitiers, Poitiers, France

²¹Department of Pediatric Hematology and Oncology, Centre Hospitalo-Universitaire de Clermont-Ferrand, Clermont-Ferrand, France

²²Department of Pediatric Hematology and Oncology, Centre Hospitalo-Universitaire de Saint Etienne, Saint-Étienne, France

²³Pediatric, Adolescent and Young Adult Oncology Department, Institut Curie Medical Center, Paris, France

²⁴Department of Paediatric and Adolescent Oncology, Gustave Roussy Cancer Campus, Villejuif, France

²⁵Department of Pediatric Hematology and Oncology, Groupe Hospitalier du Havre, Montivilliers, France

²⁶Department of Pediatric Hematology and Oncology, Centre Hospitalo-Universitaire de Brest, Brest, France

²⁷Department of Pediatric Hematology and Oncology, Centre Hospitalo-Universitaire Saint Denis de la Réunion, Saint-Denis, Réunion, France

²⁸Service National d'Onco-Hematologie Pediatrique (SNOHP), Kannerklinik, Centre Hospitalier de Luxembourg, Luxembourg City, Luxembourg

²⁹Department of Pediatric Hematology and Oncology, Cliniques Universitaires Saint Luc, Brussels, Belgium

³⁰Sorbonne Université, Institut du Cerveau-Paris Brain Institute-ICM, Inserm, CNRS, AP-HP, Hôpital Universitaire La Pitié Salpêtrière, DMU Neurosciences, Paris, France

³¹Department of Dermatology, Necker Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France

³²Department of Internal Medicine, La Pitié Salpêtrière Hospital, AP-HP, Sorbonne Université, Paris, France

³³Université Paris Cité, INSEM U976, AP-HP Pulmonary Department, Saint-Louis Hospital, Paris, France

³⁴Pathology Department, Trousseau Hospital, AP-HP, Sorbonne Université, Paris, France

³⁵Pathology Department, Necker Hospital, AP-HP, Centre-Université Paris Cité, Institut Imagine, Paris, France

## ACKNOWLEDGMENTS

The authors thank the patients and their families for their participation in this study, and all pediatricians and pathologists from the Société Française de lutte contre les Cancers de l'Enfant et de l'Adolescent (SFCE).

## FUNDING INFORMATION

This work was supported by grants from the Programme de Recherche Translationnelle en Cancérologie (grant reference 19-143), and from the Conquer Cancer Foundation ASCO Young Investigator Award funded by the Strike 3 Foundation (grant reference #11413). This project received constant, unlimited support from the Association Histiocytose France, and support from the Association pour le Recherche et l'Enseignement en Pathologie, and from the Association Recherche et Maladie Hématologiques de l'Enfant.
<figure><img src="image_10.png" /></figure>

## CONFLICT OF INTEREST STATEMENT

Ahmed Idbaih received Research grants from Carthera, Transgene, Sanofi, Air Liquide, Servier, and Nutritheragene. He is on the advisory board for Novartis, Leo Pharma, Novocure, and Boehringer Ingelheim Int. Other authors declare that they have no competing interests.

## DATA AVAILABILITY STATEMENT

The data supporting the findings of this study are available within the article and its supplementary materials. Derived data supporting the findings of this study are available from the corresponding author Sébastien Heritier on request.

## ORCID

Jean-François Emile ID https://orcid.org/0000-0002-6073-4466

Sébastien Heritier ID https://orcid.org/0000-0003-0384-6370

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## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**How to cite this article:** Hélias-Rodzewicz Z, Donadieu J, Terrones N, et al. Molecular and clinicopathologic characterization of pediatric histiocytoses. *Am J Hematol*. 2023;98(7):1058-1069. doi:10.1002/ajh.26938