MD-MSc Laura Polivka
email: laura.polivka@institutimagine.org

MD-PhD Julien Rossignol

MD-MSc Antoine Neuraz

MSc Djiba Condé

MSc Julie Agopian

MD Cécile Méni

MD-PhD Nicolas Garcelon

PhD Patrice Dubreuil

PhD Leïla Maouche-Chrétien

MD-PhD Smail Hadj-Rabia

MD-PhD Olivier Hermine

MD-PhD Christine Bodemer
email: christine.bodemer@aphp.fr

Criteria for the Regression of Pediatric Mastocytosis: A Long-Term Follow-Up

Keywords:

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Mastocytosis corresponds to a heterogeneous group of neoplastic conditions characterized by the expansion and accumulation of mast cells (MCs) in one or more organs (such as the skin, bone marrow, spleen, lymph nodes, and gastrointestinal tract). [START_REF] Arber | The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia[END_REF][START_REF] Valent | Mastocytosis: 2016 updated WHO classification and novel emerging treatment concepts[END_REF][START_REF] Hartmann | Cutaneous manifestations in patients with mastocytosis: Consensus report of the European Competence Network on Mastocytosis; the American Academy of Allergy, Asthma & Immunology; and the European Academy of Allergology and Clinical Immunology[END_REF] Disease can appear in childhood or in adulthood. Pediatric patients generally exhibit cutaneous mastocytosis (CM), in which MC infiltrates are limited to the skin and the resulting skin lesions spontaneously recede after several years in most patients. [START_REF] Méni | Paediatric mastocytosis: a systematic review of 1747 cases[END_REF][START_REF] Lange | Clinical aspects of paediatric mastocytosis: a review of 101 cases[END_REF][START_REF] Ben-Amitai | Pediatric cutaneous mastocytosis: a review of 180 patients[END_REF][START_REF] Bilgili | Comparison of cutaneous mastocytosis with onset in children and adults[END_REF][START_REF] Kiszewski | Cutaneous mastocytosis in children: a clinical analysis of 71 cases[END_REF][START_REF] Hannaford | Presentation of cutaneous mastocytosis in 173 children[END_REF][START_REF] Azaña | Urticaria pigmentosa: a review of 67 pediatric cases[END_REF] Congenital mastocytosis is defined by the presence of lesions at birth. The clinical manifestations of mastocytosis are related to the clonal accumulation of MCs (depending on which tissues are involved) and the effects of mediators released from the MCs. In fact, over 200 mediators with a role in various physiological and pathological conditions have been identified in MC granules, including heparin and proteases like tryptase and chymase. [START_REF] Mukai | Mast cells as sources of cytokines, chemokines, and growth factors[END_REF] As in cases of adult-onset mastocytosis, most of cases of pediatric mastocytosis present symptoms of mast cell activation (MCAs, due to MC mediator release), in addition to skin involvement. [START_REF] Valent | Definitions, criteria and global classification of mast cell disorders with special reference to mast cell activation syndromes: a consensus proposal[END_REF][START_REF] Valent | Proposed Diagnostic Algorithm for Patients with Suspected Mast Cell Activation Syndrome[END_REF] Somatic gain-of-function mutations in the KIT gene are found in most of patients with mastocytosis. In adult-onset mastocytosis (which is usually persistent and systemic), the KIT D816V mutation (in exon 17) is the most frequent, and is detected in more than 85% of cases. [START_REF] Longley | Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis[END_REF][START_REF] Arock | Preclinical human models and emerging therapeutics for advanced systemic mastocytosis[END_REF] In contrast, a mutation in exon 17 is found in only 43% of pediatric cases -the same prevalence as for mutations affecting exons 8, 9, 10 or 11. [START_REF] Bodemer | Pediatric mastocytosis is a clonal disease associated with D816V and other activating c-KIT mutations[END_REF][START_REF] Meni | Paediatric mastocytosis: long-term follow-up of 53 patients with whole sequencing of KIT. A prospective study[END_REF] Although it has been hypothesized that the mutational differences observed between pediatric and adult cases explain (at least in part) the regression of CM, the underlying mechanisms have not been yet identified. [START_REF] Hartmann | Cutaneous manifestations in patients with mastocytosis: Consensus report of the European Competence Network on Mastocytosis; the American Academy of Allergy, Asthma & Immunology; and the European Academy of Allergology and Clinical Immunology[END_REF][START_REF] Yanagihori | c-kit Mutations in patients with childhood-onset mastocytosis and genotype-phenotype correlation[END_REF][START_REF] Lanternier | Phenotypic and genotypic characteristics of mastocytosis according to the age of onset[END_REF] Nevertheless, a number of clinical factors appeared to be associated with spontaneous disease improvement; they include early disease onset (within 12 months of birth) and the type of lesion (e.g. maculopapular cutaneous mastocytosis (MPCM) with large lesions). [START_REF] Meni | Paediatric mastocytosis: long-term follow-up of 53 patients with whole sequencing of KIT. A prospective study[END_REF][START_REF] Wiechers | Large maculopapular cutaneous lesions are associated with favorable outcome in childhood-onset mastocytosis[END_REF] The often prominent, severe worsening of MCAs can significantly impact the patient's quality of life and can sometimes trigger life-threatening complications; however, the relationship between the regression of MCAs and the regression of skin lesions in pediatric-onset mastocytosis has not been clearly elucidated.

The objective of the present retrospective study of the largest yet cohort of well-characterized patients with pediatric-onset mastocytosis and at least 8 years of follow-up were to (i) identify the clinical, biological and molecular factors associated with spontaneous CM regression in children, and (ii) to analyze the correlation between MCA symptoms and the skin lesion regression.

Material and Methods

Study population

We performed a retrospective study in the dermatology department at Necker Children's Hospital (Paris, France), which coordinates the pediatric branch of the CEREMAST national reference center for mastocytosis. This was an observational study; laboratory tests (KIT gene sequencing and blood chemistry tests) were performed only after the patient's parents had been given a comprehensive explanation and had given their consent (see below).

Accordingly, all patients with pediatric-onset mastocytosis who had consulted or been hospitalized between January 1 st , 1996, and December 31 st , 2018, were considered for eligibility in the study (Figure 1). Mastocytosis was defined and classified according to the World Health Organization (WHO) criteria (see Supplemental Methods). In most cases, the diagnosis of CM was based solely on clear clinical signs (major criteria); for ethical reasons, a skin biopsy (to screen for minor criteria) was not performed. Patients were selected from the CEREMAST national cohort, which was set up as part of a physiopathological study of mastocytosis in which information is stored in electronic medical records. The study protocol was approved by the local investigational review board (CPPRB Groupe Hospitalier de Paris Necker", Paris, France, n°03-01-05; July 7 th ,2003). Regardless of the type of mastocytosis, KIT sequencing of a skin biopsy was always suggested if it was likely to improve the patient's care. The information extracted from the CEREMAST was supplemented with information from Necker Children's Hospital's data warehouse, which integrates free text data (hospitalization, consultation and prescription reports, etc.) and coded data (laboratory results). [START_REF] Garcelon | A clinician friendly data warehouse oriented toward narrative reports: Dr. Warehouse[END_REF] The data warehouse was queried with the following diagnostic codes: "mastocytosis", "cutaneous mastocytosis", "diffuse cutaneous mastocytosis", "mastocytoma", and "urticaria pigmentosum". We next analyzed the characteristics of individual patients by referring to their electronic medical records; erroneous diagnoses, cases of adult-onset mastocytosis, and cases of mastocytosis diagnosed less than 8 years previously were all excluded. Hence, all the included patients presented with confirmed pediatric-onset mastocytosis diagnosed at least 8 years previously. We analyzed the patient's electronic and paper-based medical records, and pictures of their skin.

Patient selection.

Between January 1 st , 1996, and December 31 st , 2018, a total of 687 patients consulted for pediatric-onset mastocytosis at the French National Reference Center. Seventyseven patients were excluded because of an incorrect diagnosis. Overall, 272 of the remaining 610 patients had suffered from pediatric-onset mastocytosis for at least 8 years, and so were selected for long-term follow-up (Figure 1). Twelve patients were lost to follow-up (the changes in CM and MCAs were not documented) and so were excluded from analyses of the regression of CM and MCAs.

Data collection. The study variables were assessed twice: at disease onset (during a consultation) and at a study visit (a 'phone interview' or a clinical examination) by dermatologists from the same department at the CEREMAST, using an electronic case report form. For all identified patients, the following items of data were collected: sex; age at onset; age at diagnosis; family history of mastocytosis; the type of cutaneous mastocytosis (MPCM, mastocytoma, or diffuse cutaneous mastocytosis (DCM)), the presence of one or more of the suggested symptoms of MCA: pruritus, flushes, urticaria, diarrhea, abdominal pain, epigastric pain, food reactions, anorexia, nausea, vomiting, loss of consciousness, hypotension, episodes of pallor, a drained feeling, asthenia, headaches, nightmares, sleep disorders, dyspnea, muscle/joint/bone pain, palpitations, polyuria, emotional disturbance or behavioral disorders, the baseline serum tryptase level, KIT gene sequencing data, and the change over time in MCAs and skin lesions. Cases of MPCM were subdivided in three groups: MPCM-large subgroup [patients with at least one large (>1 cm) or polymorphic lesion]; MPCM-small subgroup [patients with small (<5 mm) lesions] and non-classified MPCM (patients for whom the size of the lesions was not known). Congenital mastocytosis was defined by the presence of lesions at birth. The change over time in skin lesions was classified as complete regression (i.e. the healing of lesions, with the absence of Darier's sign), partial regression (a decrease in the number of lesions, and/or lightening of the lesions), or stabilization. The presence or absence of the 26 above-mentioned MCAs was rated (with one point for each symptom) at baseline and at last follow-up. The change over time in MCAs was classified as complete regression, partial regression (a decrease of at least one point), stabilization, or aggravation (an increase of at least one point).

Screening for KIT mutations

A skin biopsy was collected in 66 of 272 enrolled patient for KIT mutation analysis using a highly sensitive technique (described in the Supplemental Methods) but only if the patient's parents or legal guardian had given his/her informed consent for genetic screening or if the patient had systemic mastocytosis (SM) requiring specific treatment. A bone marrow biopsy with KIT sequencing was performed only in patients (n=7) with signs suggestive of systemic mastocytosis (cytopenia, organomegaly, malabsorption syndrome and bone damage). Three categories of KIT mutation were defined: a point mutation at codon 816, a mutation outside codon 816, and the wild type (WT).

Statistical analyses are described in the Supplemental Methods.

Results

Characteristics of the study population

The main characteristics of the study population (n=272) are summarized in Table 1. The male-to-female ratio was 1.23, 115 patients (42.3%) had congenital mastocytosis. Twentythree patients (8.5%) had familial mastocytosis (i.e. at least one first-degree parent with mastocytosis). The mean (range) disease duration at the end of our study was 12.93 years (8.1-31.7). We observed 211 (77.6%) patients with MPCM, 46 (16.9%) with mastocytoma, and 15 (5.5%) with DCM. The frequency of congenital forms was higher among patients with mastocytoma (54.3%) or DCM (53.3%) than among patients with MPCM (38.9%), although the intergroup differences were not statistically significant (p=0.069 and p=0.29, respectively). One hundred and ninety-five patients (71.7%) presented with MCAs at diagnosis, with a mean (range) of 2.04 signs (1-11). There were no significant differences between the MPCM, mastocytoma, and DCM groups with regard to the number of MCAs at baseline, except that cutaneous signs of MCA (pruritus and/or flushes) were significantly more frequent in the DCM group (73.3%) than in the MPCM group (40.8%; p=0.016, Table 2 and Figure E1). The KIT gene was sequenced (using a skin biopsy sample) in 66 of the 272 patients (24.3%): 28 of these 66 (42.4%) carried a mutation in codon 816 (exon 17), 28 (42.4%) carried a KIT mutation outside codon 816, and 10 (15.2%) had a WT KIT gene. Fifty of the 66 patients have already been described by Bodemer et al. [START_REF] Bodemer | Pediatric mastocytosis is a clonal disease associated with D816V and other activating c-KIT mutations[END_REF] The different types of KIT mutation are shown in Table E1. No genotype-phenotype correlations were apparent (data not shown). The mean serum tryptase level at baseline was 7.52 µg.L -1 [1-60] in the 121 assayed patients. As expected, the mean (range) tryptase level was significantly higher in the DCM group [23.99 (2-60) µg.L -1 ] than in the MPCM group [7.36 (1 -45.2) µg.L -1 ; p<0.0001] and significantly higher in the MPCM group than in the mastocytoma group [4.32 µg.L -1 , (1.3-11.5), p=0.045] (Figure E1). Apart from the tryptase level, the MPCM, mastocytoma and DCM groups did not differ significantly (data not shown) with regard to the main characteristics summarized in Table 1.

Within the MPCM group, 85 patients (40.3%) had large MPCM lesions, 78 (37%) had small MPCM lesions and 48 patients (22.7%) had non-classified MPCM lesions. The frequency of familial mastocytosis was significantly higher in the MPCM-small subgroup (17.9%) than in the MPCM-large subgroup (5.9%, p=0.026), whereas the frequency of congenital mastocytosis was higher in the MPCM-large group (48.2% vs. 37.5%, p=0.016). Disease onset after the age of 2 was recorded for 19 patients, all of whom belonged to the MPCMsmall or -non-classified subgroups. The groups did not differ significantly with regard to the distribution of the KIT mutations (Table 1). The characteristics of the included participants and the screened (source) population were similar (n=610, Table E2). Five of the 610 patients in the source population presented with SM (as defined by the WHO criteria): three of these five had KIT-D816V-positive, congenital, aggressive systemic mastocytosis (ASM) from the onset, with DCM and MC infiltration of the bone marrow, liver, spleen and digestive tract; the remaining two developed acute myeloid leukemia in which a t(8;21) or (1;17) translocation was associated with a KIT D816V or N822Y mutation, respectively (Table E3). [START_REF] Arber | The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia[END_REF] The main differences between patients for whom mastocytosis was congenital or appeared within one month of birth (the congenital group) and those for whom mastocytosis appeared later than a month after birth (the non-congenital group) are summarized in Table 3. We found that MPCM was significantly more common in the non-congenital group (83.4%) than in the congenital group (70.9%, p=0.019). Mastocytoma and DCM were more common (but not significantly) in the congenital group. The distribution of KIT mutations (notably the frequency of KIT point mutations in codon 816) was similar in the two groups (Figure E2).

The distribution of the KIT mutations as a function of age of onset is shown in Figure E2.

Changes over time in CM

Two hundred and ten of the 260 patients (80.8%) showed complete (n=18) or partial (n=192) regression of CM at last follow-up (Figure E3). The mean (range) time to regression was 6.1 years (1.48-21.9). Forty-one patients were stable at the last examination, and 9 patients had a greater number of lesions (i.e. worsening). The clinical, biological and molecular variables associated with changes in CM are summarized in Table 4. MCAs from the age of 1 developed a small but painful osteolytic bone lesion with MC infiltrates at puberty but did not meet the criteria for ASM. Another KIT D816Y+ patient with MPCM-large congenital mastocytosis developed hepatomegaly. No liver biopsies were performed. In both cases, a bone marrow aspirate confirmed the presence of MC infiltrates.

None of the 272 included patients had developed ASM.

Changes over time in MCA symptoms

Seventy-seven patients (29.6%) were free of MCAs throughout the study. Of the remaining 183 patients, 144 (78.7%) showed complete (n=85) or partial (n=59) regression of the MCAs.

Twenty-two (12%) patients were classified as stable at the last examination, and the number of MCAs increased in 17 (9.3%) patients (i.e. worsening). The clinical, biological and molecular markers associated with an increase in the number of MCAs are summarized in Table 5. The number of MCAs (and especially the presence of urticaria) at baseline was a prognostic factor for the worsening of MCAs (OR=653, 95%CI=[1.18.10 2 -1.24.10 4 ], p=0.000 and OR=4.97, 95%CI=[1.05-26.2], p=0.041, respectively). Taken individually, the other types of MCAs (digestive symptoms, pain, a drained feeling etc.) were not significantly associated with worsening of MCAs (data not shown). Likewise, sex, congenital mastocytosis, familial mastocytosis, the type of CM, the different types of MPCM and the type of KIT mutation were not significantly associated with the course of the MCAs. The serum tryptase level at baseline was not significantly associated with the course of the MCAs (OR=1.05, 95%CI=[0.973 -1.17], p=0.341). In a multivariate analysis, however, a KIT point mutation in exon 816 became significantly associated with the regression of MCAs (Table E4).

The correlation between changes in MCAs and CM

Aggravation/stability of MCAs was significantly correlated with worsening/stability of CM (OR=3.36, p=0.015). However, baseline MCAs persisted or even worsened in 27 patients with a favorable CM outcome (Figure 2). The regression of both MCA symptoms and CM was associated with the number of MCAs at baseline and the presence of cutaneous symptoms (pruritus and flushing), relative to patients not showing this correlation (OR=2.5 and 1.97, respectively, p=0.0002, Table 6).

Cluster analysis of the different MCAs at diagnosis and changes in CM/MCAs

A cluster analysis of the different MCAs identified four clusters of patients (Figure E4). Clinically, cluster 1 was characterized by isolated food intolerance, cluster 2 was not highly specific, cluster 3 was characterized mainly by flushes and pruritus, fatigue with a drained feeling, abdominal pain and intestinal transit disorders, and cluster 4 was characterized by pruritus (but not flushes), abdominal pain, and intestinal transit disorders. The changes over time in each group are depicted in Figure E4. There were no significant intergroup differences with regard to changes over time in CM. Patients in cluster 3 were significantly more likely to experience a decrease in MCAs outcome than patients in cluster 1 (taken as a reference) were (p=0.008, Figure E4). The same association was seen for cluster 4 but was not statistically significant (p=0.08).

Discussion:

Here, we analyzed a large cohort of patients with pediatric-onset mastocytosis (n=610) and defined prognostic factors in 260 patients with at least 8 years of follow-up (mean duration of disease: 12.93 years). We showed that a spontaneous CM regression was significantly associated with not only congenital disease but also (unexpectedly) a KIT D816V mutation. Moreover, even though the worsening of MCAs was correlated with the worsening of CM, MCAs continued to worsen into adulthood in a subset of patients with CM regression. Sex and familial mastocytosis were not associated with changes in CM.

The demographic characteristics of our study population were similar to those reported in the literature, namely slight male predominance (male/female ratio: 1.23), disease onset early in life (mean age at onset: 301.2 days), a relatively high frequency of congenital forms (42.3%), a predominance of MPCM (77.6%) relative to mastocytoma (16.9 %) and DCM (5.5%), and a mean time to CM regression of 6.1 years (Table 1). [START_REF] Méni | Paediatric mastocytosis: a systematic review of 1747 cases[END_REF] Although aggressive forms of SM were rare (n=5 out of 610, 0.82%), the fact that they were always clinically apparent at disease onset meant that we did not systematically have to obtain bone marrow aspirates in children lacking obvious clinical signs of ASM (organomegaly with impaired organ function, pancytopenia, etc.).

As expected, a large minority (42.4%) of the patients presented with a KIT point mutation in codon 816. [START_REF] Bodemer | Pediatric mastocytosis is a clonal disease associated with D816V and other activating c-KIT mutations[END_REF] The present report is the first to show that this mutation is (like congenital CM) significantly associated with CM regression (OR=0.173, p=0.031, Table 4) in pediatric-onset mastocytosis; however, in this study we confirmed the presence of the KIT D816V mutation in only 28 patients. In contrast, the KIT D816V mutation is associated with disease persistence in adult forms of mastocytosis. [START_REF] Arock | KIT mutation analysis in mast cell neoplasms: recommendations of the European Competence Network on Mastocytosis[END_REF] In our study, mutations outside codon 816 did not appear to be significantly associated with the change over time in CM; this was probably because the follow-up period was too short.

We did not find a KIT mutation in two patients with worsened of cutaneous lesions. As in adult forms of ASM, the absence of a KIT mutation suggests the causal presence of one of more other gene mutations. In the present study, the absence of a KIT mutation appeared to be associated with the worsening of CM (OR=3.0, 95%CI=[0.666 -12.6], p=0.135, Table 4). [START_REF] Molderings | The genetic basis of mast cell activation disease -looking through a glass darkly[END_REF] The distribution of the KIT mutations (notably the KIT point mutation in codon 816) was similar in congenital and non-congenital forms of mastocytosis (Figure E2). Thus, the mutational differences between children and adults with mastocytosis (especially the high prevalence of KIT D816V mutation in adults) cannot alone explain the outcome of CM. Based on Gentek et al.'s research on the ontogeny of MCs, we propose a new hypothesis for the regression of pediatric-onset mastocytosis. [START_REF] Gentek | Hemogenic Endothelial Fate Mapping Reveals Dual Developmental Origin of Mast Cells[END_REF] Gentek et al. showed that in the embryonic mouse, MCs have two hematopoietic origins: the yolk sac and the bone marrow. The two MC lineages differ with regard to the phenotype and the gene expression pattern. Interestingly, embryonic skin MCs derived from the yolk sac persist after birth and are then gradually replaced by the definitive adult MCs. We therefore hypothesized that regardless of the nature of the KIT gene mutation, mastocytosis will regress over time if the mutation occurs in senescent yolk-sac MCs. Here, we did not find a correlation between the KIT genotype and the patient phenotype; hence, the KIT mutation alone does not explain the heterogeneous symptoms observed in all the various kinds of mastocytosis. This observation strongly suggests that one or more mutations in one or several genes are involved -perhaps in synergy with a KIT mutation -in the development of mastocytosis. [START_REF] Zappulla | Mastocytosis in mice expressing human Kit receptor with the activating Asp816Val mutation[END_REF][START_REF] Mayerhofer | Unique effects of KIT D816V in BaF3 cells: induction of cluster formation, histamine synthesis, and early mast cell differentiation antigens[END_REF] The association between KIT mutation and regression may be related to the presence of other beneficial factors such as an increase over time in the expression of MC-inhibiting chondroitin or heparan sulfate. [START_REF] Gross | Chondroitin sulfate inhibits secretion of TNF and CXCL8 from human mast cells stimulated by IL-33[END_REF] The aggravation of MCAs was significantly correlated with the worsening of CM (OR=3.36, p=0.015, Table 4). Interestingly, MCAs persisted or even worsened in 27 of the 142 patients (19%) with these symptoms at baseline and a favorable CM outcome (Figure 2) -suggesting that long-term follow-up of these patients might nevertheless be necessary. Thus, we identified two subgroups of patients with CM regression: (i) those in whom both CM and MCAs regressed (n=115), and (ii) those in whom CM regressed despite the worsening or persistence of MCAs (n=27, subgroup 2). The only significant differences between these two groups were the number of MCAs at baseline and the presence of skin symptoms (pruritus and flushing). Surprisingly, pruritus and flushing were more frequent in the "CM and MCA regression" subgroup than in the other subgroup (number of MCAs: 2.5 and 1.97 respectively, p=0.0002, Table 6) -probably because these symptom;s are more strongly associated with the abnormally high MC count than with MC activation per se. The difference between these groups might be related to the presence of mutations or polymorphisms in one or more of the genes involved in MCA; however, addressing this question would require an extensive molecular investigations. The evolution of the levels of serum tryptase, and urinary n-methyl-histamine, urinary 2,3-dinor-11β-prostaglandin F2α, and urinary leukotriene E4 in this patient population might provide further insights.

The present study is the first to have reported on such a large number of cases and to have specified the various MCAs and their changes over time. Symptoms of MCA were frequent in patients with CM (71.7%) and appeared not to be related to the extent of skin involvement (except for pruritus and flushing, Table 2 and Figure E1). Thus, the type and the number of MCAs appear to be more strongly related to a qualitative defect in diseased MCs than to the high MC count per se. Of all the individual MCAs, only the presence of urticaria at diagnosis was significantly associated with a worsening of MCAs in general. At present, clinicians lack a validated scoring system for the qualitative and quantitative evaluation MCAs in children.

The development of this type of score might improve patient management in general and the assessment of drug efficacy in particular.

Our cluster analysis of the MCAs at baseline identified between four clusters of patients (Figure E4). In two clusters (patients presenting with pruritus and flushes at baseline or pruritus without flushes at baseline, respectively), the MCAs decreased over time. These findings will have to be validated in a study with greater statistical power.

Although our sample size was large for a rare disease, the study's retrospective design, and the low percentage of patients with KIT gene sequence data constituted limitations.

Conclusion

In a retrospective study of the largest yet cohort of well-characterized patients with pediatriconset mastocytosis and at least 8 years of follow-up by the same clinicians, we showed that congenital mastocytosis and the KIT D816V mutation were associated with spontaneous CM regression. Furthermore, even though the worsening of MCAs was correlated with the worsening of CM, we found that MCAs continued to worsen into adulthood in a subset of patients with CM regression. The latter observation suggests that several different mechanisms are involved in the accumulation and the activation of MCs and justifies longterm follow-up in these pediatric patients with CM regression.

  Congenital mastocytosis was associated with CM regression (odds ratio (OR)=0.476, 95% confidence interval (CI)=[0.236-0.917], p=0.031). The presence of one or more MCAs at baseline was a significant prognostic factor for CM progression (OR=2.20, 95%CI=[1.05 -5.06], p=0.047). However, each individual MCAs (digestive symptoms, pain, a drained feeling etc.) was not significantly associated with changes over time in CM (data not shown), except for the presence of pruritus or flushes at baseline; surprisingly, these were associated with CM regression (OR=0.19, 95%CI=[0.072-0.43], p=0.000; and OR=0.3, 95%CI=[0.128 -0.663], p=0.004, respectively). Aggravation or stabilization of MCAs was significantly correlated with worsening CM (OR=3.36, 95%CI=[1.28 -9.13], p=0.015). Sex, familial mastocytosis, the type of CM, the different types of MPCM and the serum tryptase level at baseline were not significantly associated with the disease outcome. The presence of a KIT point mutation in codon 816 was significantly associated with CM regression (OR=0.173, 95%CI=[0.025-0.719], p=0.031). In contrast, a WT KIT genotype was associated (albeit not significantly) with the aggravation of skin lesions (OR=3, 95%CI=[0.666-12.6], p=0.135). Of the 50 stable (n=41) or worsening (n=9) patients, 14 patients (12 stable and 2 worsening) had KIT sequence data: two patients presented with a KIT point mutation at codon 816, eight patients presented with other KIT mutations, and four patients were WT (notably two patients with worsening). Two patients developed extracutaneous disease features. A KIT D816V+patient in the MPCM-small subgroup with

Figure 1 .

 1 Figure 1. Study flow chart

Figure 2 .

 2 Figure 2. Correlation between CM regression and changes over time in MCAs. CM: cutaneous mastocytosis, MCAs: mast cell activation symptoms, MPCM: maculopapular cutaneous mastocytosis.

Figure 1 .Figure 2 .

 12 Figure 1. Study flow chart

Table and figure legendsTable 1 .

 1 Main characteristics of the study population and the different cutaneous mastocytosis subtype groups.

	DCM: diffuse cutaneous mastocytosis, F: female, M: male, MCAs: mast cell activation
	symptoms, MPCM: maculopapular cutaneous mastocytosis, NC: non-classified.
	*p<.05

Table 2 .

 2 Main mast cell activation symptoms at baseline, as a function of the type of

	cutaneous mastocytosis
	DCM: diffuse cutaneous mastocytosis, MPCM: maculopapular cutaneous mastocytosis.
	*p<.05

Table 3 .

 3 Congenital vs. non-congenital mastocytosis DCM: diffuse cutaneous mastocytosis, F: female, M: male, MPCM: maculopapular cutaneous mastocytosis, NC: non-classified.

	*p<.05

Table 4 .

 4 Univariate analysis -clinical, biological and molecular variables associated with the

	progression of cutaneous mastocytosis
	MCAs: mast cell activation symptoms, MPCM: maculopapular cutaneous mastocytosis.
	*p<.05, **p<.01, ***p<.001
	1 Other types of MCAs (digestive symptoms, pain, a drained feeling etc.) were not
	significantly associated with changes in CM (data not shown)

Table 5 .

 5 Univariate analysis -clinical, biological and molecular variables associated with the

	progression of MCAs.
	CM: cutaneous mastocytosis, MCAs: mast cell activation symptoms, MPCM: maculopapular
	cutaneous mastocytosis.
	*p<.05, ***p<.001

Table 6 .

 6 A comparison of clinical, biological and molecular variables in patient group 1 (correlated regression of both CM and MCAs) and patient group 2 (no correlation).

Table 1 .

 1 Main characteristics of the study population and the different cutaneous mastocytosis subtype groups

	Characteristics Population	MPCM	MPCM-	MPCM-	p.value	MPCM-	Mastocytoma	DCM
		n = 272	n = 211	large	small	(MPCM-	NC	n = 46	n = 15
		(n/total%)	(n/total%)	n = 85	n = 78	large vs	n = 48	(n/total%)	(n/total%
						MPCM-			)
						small)
	Clinical
	Females	122	93	37	36	-	20	22	7
	Males	150	118	48	42	-	28	24	8
	Sex ratio (M/F)	1.23	1.27	1.43	1.17	0.75	1.4	1.09	1.14
	Mean age at	1308.5	1319	1369.3	1348.6	-	1184.5	1263,2	1302
	diagnosis (days)
	Mean age at	301.23	302.22	305.93	302.22	-	305.05	300,87	329,82
	onset (days)
	Congenital	115 (42.3)	82 (38.9)	41	23	0.016*	18	25 (54.3)	8 (53.3)
				(48.2)	(29.5)		(37.5)
	Familial	23 (8.5)	22 (10.4)	5 (5.9)	14	0.026*	3 (6.25)	1 (2.2)	0 (0)
	mastocytosis				(17.9)
	Number of	2.04	2.09	2.09	2.19	0.75	1.52	1.85	1.93
	MCAs at
	diagnosis (mean)
	Absence of	77 (28.3)	59 (28)	23	15	0,27	21	16 (34.8)	2 ( (13.3)
	MCAs			(27.1)	(19.2)		(43.8)
	Molecular -	66	58	23	26		9	4	4
	Sequencing of
	KIT gene
	KIT point	28 (42.4)	28 (48.3)	11	12	1	5 (55.6)	0 (0)	0 (0)
	mutation at			(47.8)	(46.2)
	codon 816
	Other KIT	28 (42.4)	22 (37.9)	9 (39.2)	10	1	3 (33.3)	3 (75)	3 (75)
	mutations				(38.5)
	KIT WT	10 (15.2)	8 (13.8)	3 (13)	4 (15.4)	1	1 (11.1)	1 (25)	1 (25)
	Biological
	Level of	7.52 [1 -	7.36 [1-	7.23	7.55 [1-	0.82	7.60	4.32 [1.3 -	23.99 [2-
	baseline tryptase	60]	45.2]	[1.5-	45.2]		[2.9-	11.5]	60]
	[mean (µg.L -1 ),	(n=121)	(n=101)	23.1]	(n=42)		18.3]	(n=16)	(n=4)
	range, number of			(n=48)			(n=11)
	patients]

Table 2 .

 2 Main mast cell activation symptoms at baseline, as a function of the type of cutaneous mastocytosis

		MPCM (n/total %) n = 211	Mastocytoma (n/total %) n = 46	DCM (n/total %) n = 15	p.value (MPCM vs Mastocytoma)	p.value (MPCM vs DCM)
	Cutaneous signs:
	Pruritus/Flushes	86 (40.8)	21 (45.7)	11 (73.3)	0.62	0.016*
	Urticaria	5 (2.4)	1 (2.2)	1 (6.7)	1	0.34
	Digestive symptoms: anorexia,
	abdominal pain, diarrhea ,	72 (34.1)	11 (23.9)	4 (26.7)	0.22	0.78
	nausea and /or vomiting
	Food reactions	32 (15.2)	7 (15.2)	1 (6.7)	1	0.70
	Loss of consciousness, drained
	feeling and/or episodes of	44 (20.9)	7 (15.2)	2 (13.3)	0.54	0.74
	pallor
	Sleep disorders and/or nightmares	17 (8.1)	3 (6.5)	1 (6.7)	1	1
	Muscle/joint/bone pain(s)	18 (8.5)	3 (6.5)	2 (13.3)	1	0.63
	Emotional disturbance or behavioral disorders	2 (0.9)	1 (2.2)	0 (0)	0.45	1
	Pollakiuria	5 (2.4)	1 (2.2)	0 (0)	1	1

Table 3 .

 3 Congenital vs. non-congenital mastocytosis

	Characteristics	Congenital group	Non-congenital group	p.value
		n = 127 (n/total %)	n = 145 (n/total %)
	Clinical
	Females	52 (40.9)	70 (48.3)
	Males	75 (59.1)	75 (51.7)	0.271
	Sex ratio (M/F)	1.4	1.07
	Mean age at diagnosis	1237.3	1371.3	0.544 (t test)
	(days)
	Familial mastocytosis	8 (6.3)	15 (10.3)	0.28
	MPCM	90 (70.9)	121 (83.4)	0.019 *
	-Large (polymorphic	44 (48.9)	41 (33.9)	0.033*
	lesions)
	-Small (<0.5cm)	27 (30)	51 (42.1)	0.084
	-NC	19 (21.1)	29 (24)	0.34
	Mastocytoma	27 (21.3)	19 (12.9)	0.26
	DCM	10 (7.9)	5 (3.4)	0.12
	Molecular -	29 (22.8)	37 (25.5)
	Sequencing of KIT gene
	KIT point mutation at	12 (41.4)	16 (43.2)	0.93
	codon 816
	Other KIT mutations	13 (44.8)	15 (40.5)	0.804
	KIT WT	4 (13.8)	6 (16.2)	1
	Biological
	Level of baseline	8.92 [1-60]	6.53 [1.1-28.2]
	tryptase [mean (µg.L -1 ),	(n=52)	(n=69)
	range, number of
	patients]

Table 6 .

 6 A comparison of clinical, biological and molecular markers in patient group 1 (correlated regression of both CM and MCAs) and patient group 2 (no correlation)

	N = 142 patients with CM	Groupe 1	Groupe 2	p.value
	regression and changes in MCAs	n= 115 (n/total %)	n = 27 (n/total %)
	Clinical
	Sex ratio (M/F)	1.25	1.45	0.83
	Familial mastocytosis	14 (12.2)	1 (3.7)	0.0097
	Congenital mastocytosis	48 (41.7)	12 (44)	0.34
	MPCM	89 (77.4)	23 (85.2)	0.44
	Large	38	6	0.16
	Small	37	10	1
	NC	14	7	0.13
	Mastocytoma	20 (17.4)	3 (13)	0.57
	DCM	6 (5.2)	1 (3.7)	1
	Number of MCAs at diagnosis	2.5 [1-11]	1,97 [0 -4]	0.0002***
	Cutaneous signs:	81 (70.4)	12 (44.4)	0.014*
	Pruritus/Flushes/Urticaria
	Digestive symptoms:	58 (50.4)	10 (37.0)	0.28
	anorexia, abdominal pain,
	diarrhea, nausea and /or
	vomiting
	Discomfort, loss of	35 (30.4)	4 (14.8)	0.15
	consciousness, drained
	feeling and/or episodes
	of pallor
	Food reaction	28 (24.3)	4 (14.8)	0.44
	Bone, joint and/or	15 (13)	0 (0)	0.074
	muscular pain(s)
	Biological
	Level of baseline tryptase [mean	7.54 [1 -45.2] (n=64)	7.75 [1.5-28.2] (n=11)	0.379
	(µg.L -1 ), range, number of
	patients]
	Molecular -Sequencing of KIT	n=36	n=6	-
	gene
	Other KIT mutations	13 (36.1)	3 (50)	-
	KIT point mutation at codon 816	18 (50)	3 (50)	-
	KIT WT	5 (13.4)	0 (0)	-

Other types of MCAs (digestive symptoms, pain, a drained feeling etc.) were not

Other types of MCAs (digestive symptoms, pain, drained feeling etc.) were not significantly associated with the overall progression of MCAs (data not shown)

Acknowledgements

The authors acknowledge the contribution and participation of the pediatric mastocytosis study group: Professor Sébastien Barbarot, Dr. Nathalie Beneton, Dr. Benoît Catteau, Dr. Karine Despontin, Dr. Jean Donadieu, Dr. Catherine Eschard, Dr. Marion Gillibert-Yvert, Dr. Anne Hoppe, Dr. Catherine Humiliere, Professor Gisele Kanny, Dr. Laurence Lion, Professor Ludovic Martin, Professor Annabel Maruani, Dr. Guy Molitor, Dr. Alice Phan, Dr. Jean-Louis Salomon, Dr. Beatrice Sans, Dr. Francois Skowron, Dr. Jean-Francois Stadler, Dr. Christine Labreze, Dr. Stéphane Barete, Dr. Benoit Catteau, Dr. François Skowron and Dr.

Yves Turc (data collection). Dr. Laura Polivka was funded by the Bettencourt Schueller Foundation.

This research did not receive any specific funding from agencies or organizations in the public, commercial, or not-for-profit sectors.

Abbreviations