Clinical study of MAP2K1-mutated Langerhans cell histiocytosis in children

To analyze the genetic and clinical features of children with MAP2K1-mutated Langerhans cell histiocytosis (LCH). We compared the clinical features of 37 children with MAP2K1-mutated LCH with those of the BRAFV600E mutation group (n = 133) and no known mutation group (n = 59) in the same period. We found 13 mutations of the MAP2K1 gene, which were mainly concentrated at p.53–62 and p.98–103. The most common mutation site was c.172_186del (12/37). Compared with the BRAFV600E mutation group, the patients with MAP2K1 mutations were mainly characterized by single-system multiple bone involvement (P = 0.022), with later disease onset (P = 0.029) as well as less involvement of risk organs, especially liver (P = 0.024). There was no significant difference in clinical features compared with the no known mutation group. The 2-year progression-free survival rate of first-line treatment (ChiCTR1900025783, 07/09/2019) in MAP2K1-mutated patients was 65.6% ± 9.5%. The prognosis of patients with lung involvement was poor [HR (95% CI) = 6.312 (1.769–22.526), P = 0.005]. More progression or relapses could be found in patients with bony thorax involvement (8/17 vs. 2/20, P = 0.023), yet involvements in other sites of bones, such as craniofacial bone involvement (8/26 vs. 2/11, P = 0.688) and limb bone involvement (5/12 vs. 5/25, P = 0.240), were not correlated to disease progression or relapse. The children with MAP2K1-mutated LCH have specific clinical features requiring clinical stratification and precise treatment. MAP2K1-mutated patients with lung involvement (especially with bony thorax involvement) had poor prognosis.


Introduction
Langerhans cell histiocytosis (LCH) is a rare clonal disease characterized by the expansion of Langerhans cells derived from myeloid precursors and is more common in children than in adults (Allen et al. 2018). Various clinical manifestations of LCH can be found with many systems involved, such as bone, skin, lung, liver, spleen, etc. Among them, the prognosis of patients with risk organ (RO) involvement, including the liver, spleen and hematopoietic system, is generally poor. The risk of sequelae of the central nervous system (CNS) and disease reactivation is higher in patients with CNS-risk lesions (including craniofacial bone, eye, ear and oral involvement) (Chow et al. 2017).
At present, some studies have found that there is a clear correlation between the MAPK pathway and the pathogenesis of LCH. BRAF V600E is the most common gene mutation, identified in more than 60% of LCH patients. MAP2K1 is another common mutant gene whose mutation frequency varies from 8.6 to 46% (Lian et al. 2019). The activated coding product of the MAP2K1 gene, MAP2K1 kinase, can activate its downstream ERK kinase, which in turn affects the proliferation, differentiation and other functions of cells (Hayase et al. 2020).
In this study, the gene mutation sites, clinical features and prognosis of patients with the BRAF V600E mutation or no known mutation of the MAPK pathway were compared with that of newly treated patients with MAP2K1-mutated LCH to provide evidence for the clinical diagnosis and treatment of MAP2K1-mutated LCH.

MAP2K1 mutation group
This was a single-center retrospective case-control study. All children newly diagnosed with MAP2K1-mutated LCH who were admitted to Beijing Children's Hospital, Capital Medical University, between July 1, 2017, and October 31, 2020, were enrolled. The inclusion criteria were as follows: (1) the age of disease onset was less than 18 years old; (2) diagnosed with LCH according to the pathological examinations of biopsied tissue: positive staining of the lesion cells with CD1a and/or CD207 (Langerin), or Birbeck granules could be found under an electron microscope; (3) MAP2K1 mutation was detected in biopsied tissue before treatment; (4) newly treated patients; (5) accepted regular follow-up and evaluation of treatment response on time. The exclusion criteria were as follows: (1) the clinical data at the time of initial diagnosis could not be obtained, and (2) they had received chemotherapy or targeted drug treatment before enrolment or did not follow the treatment regimen of our center.

BRAF V600E mutation group and no known mutation of the MAPK pathway group
In the same period, all LCH patients with the BRAF V600E mutation or no known mutation of the MAPK pathway in biopsied tissue were included as the control groups. Except for the gene mutation, the other inclusion and exclusion criteria were the same as those for the MAP2K1 mutation group.

First-line treatment
Before September 2019, all patients diagnosed with LCH were treated with the LCH-III regimen developed by Histiocyte Society (HS) (Histiocyte Society. 2009) except that methotrexate was not used in this regimen, and those diagnosed after September 2019 were treated with the CCHG-LCH-2019 regimen of our center (ChiCTR1900025783), in which patients were treated with the same dosage and course of prednisone as the LCH-2009 regimen combined with vincristine 1.5 mg/m 2 , vindesine 3 mg/m 2 or vinblastine 6 mg/m 2 . Patients with isolated disease located in functionally critical anatomical sites, such as vertebral lesions with intraspinal soft tissue extension or craniofacial bone involvement with intracranial soft tissue extension, were also treated with systemic therapy (Histiocyte Society. 2009). The treatment response was evaluated at the 6th, 12th, 25th and 52nd weeks during chemotherapy and at the 3rd month, 6th month, 1st year, 2nd year and 3rd year after drug withdrawal.

Second-line treatment
The patients who met one of the following criteria were given second-line treatment: (1) the patients without RO involvement (RO − ) who were evaluated as having disease progression (active disease-worse, AD-Worse) at the 6th week of first-line chemotherapy; or were evaluated as AD-Worse or intermediate response (active disease-mixed, AD-Mixed) at the 12th week of first-line chemotherapy; (2) patients with pulmonary or pituitary involvement who had no improvement of pulmonary and pituitary lesions at the 6th or 12th week of first-line chemotherapy compared with the previous response assessment; (3) patients with RO 1 3 involvement (RO + ) who had no significant improvement in risk organs (the enlarged part of the liver and spleen reduced less than half, or there was still primary disease-related fever or hematocytopenia), primary disease-related organ dysfunction, or whose overall response was evaluated as AD-Worse or AD-Mixed at the 6th and 12th week of firstline chemotherapy; and (4) the lesions of the risk organs, CNS-risk sites, CNS system or lung were aggravated during maintenance chemotherapy or after drug withdrawal. Before September 2019, patients received HS-LCH salvage treatment if they met one of the criteria above; after September 2019, patients were treated with second-line chemotherapy with CCHG-LCH-2019 (cytarabine ± cladribine, with the same dosages and courses as HS-LCH salvage treatment). The treatment response was evaluated every four courses during second-line chemotherapy. The evaluation after withdrawal was the same as that with first-line chemotherapy.

Follow-up
Each evaluation was conducted by ultrasound (abdominal organs, thyroid gland, lymph nodes and soft tissue), X-ray film (limb bones), computed tomography (lung, skull bone) and magnetic resonance imaging (pituitary, brain and spinal marrow), according to the Histiocyte Society Evaluation and Treatment Guidelines (2009). Follow-up was continued until December 1, 2020, or until the patient died.

Determination of mutations in biopsied tissue
Genomic DNA was extracted from 10 × 5 μm unstained sections of formalin-fixed paraffin-embedded tissue using the QIAamp DNA formalin-fixed paraffin-embedded Tissue Kit (Qiagen, Hilden, Germany). Next-generation sequencing of whole exomes was used to detect mutations in the MAPK pathway (using the HiSeq 4000 sequencing platform before 2018 and the Illumina NovaSeq 6000 sequencing platform after 2018). The genomic DNA of oral mucosal cells was taken as a normal control from 61 patients, and the remaining patients had only biopsied tissues sampled.

Cytokine measurement
At initial diagnosis, serum was separated from 2 mL non-anticoagulated peripheral blood by centrifugation at 3000 rpm for 10 min. The levels of IFN-γ, TNF-α, IL-10, and IL-6 in serum were measured by FACSCalibur (BD, USA) with human Th1/Th2 cytokine detection kit (Jiangxi Cellgene Biotech Co., LTD, China) according to the manufacturer's instruction.

Statistical analysis
IBM SPSS (23.0) software was used for the statistical analysis. The measurement of data with normal distribution was expressed as mean ± SD, while the measurement of data with nonnormal distribution was expressed as the median (upper and lower quartile). For continuous variables, data were analyzed using the t test and Mann-Whitney U test, depending on the data distribution. The count data were expressed as the number of cases or percentages, and the chi-square test was used for the categorical variables. The objective response rate (ORR) after 6 weeks of first-line chemotherapy was defined as the percentage of patients with nonactive disease (NAD) and AD-Better among all patients at the 6-week evaluation of first-line chemotherapy, and the disease control rate (DCR) after 6 weeks of first-line chemotherapy was defined as the percentage of patients with NAD, AD-Better and AD-Stable among all patients at the 6-week evaluation of first-line chemotherapy. Progression-free survival (PFS) was estimated from the date of diagnosis to the date of one of the following events: AD-Mixed or AD-Worse; relapse or reactivation after drug withdrawal or death, whichever came first; the last contact with the patient. Survival curves were estimated using the Kaplan-Meier method, and the log-rank test was used to estimate the differences among the groups. A Cox proportional hazards model was used to analyze the independent prognostic factors of the MAP2K1 mutation group. A P value of < 0.05 was considered statistically significant.
In 81.1% (30/37) of the patients, the mutations were concentrated in amino acids 53-62, which were located in the negative regulatory region of MAP2K1 kinase; in the other 7 cases (18.9%), the mutations were concentrated in amino acids 98-103, which were located in the catalytic core of MAP2K1 kinase. There were two point mutations in one patient, p.I103N and p.Y130C; the latter was not reported.

Clinical and laboratory characteristics of patients with MAP2K1 mutation
The male-to-female ratio of the 37 patients with a MAP2K1 mutation was 1.31:1, and the median age at disease onset was 4.56 (1.54, 9.42) years old. The median follow-up time was 17.67 (4.82, 24.92) months. The BRAF V600E mutation group and no known mutation of the MAPK pathway group included 133 patients and 59 patients, respectively. There were more boys than girls in the three groups, but there was no significant difference in the sex ratio among the groups ( Table 2). The age at disease onset in the MAP2K1 mutation group was higher than in the BRAF V600E mutation group (Z = -2.179, P = 0.029, Table 2), but there was no difference compared with the no known mutation group (P > 0.05, Table 2).
A significantly lower incidence of multisystem (MS) involvement (with or without RO involvement, MS-RO + / MS-RO − ) and a higher incidence of single-system (SS) involvement were found in the MAP2K1 mutation group than in the BRAF V600E mutation group (χ 2 = 5.892, P = 0.015). Only one MAP2K1-mutated patient (2.7%) had RO involvement, with hemophagocytic lymphohistiocytosis (HLH). The incidence of RO involvement was significantly lower than that of the BRAF V600E mutation group (χ 2 = 7.653, P = 0.006), especially in liver involvement (2.7% vs. 17.3%, P = 0.024). In addition, more patients with SS-multiple bone involvement (48.6% vs. 28.6%, P = 0.022) and fewer patients with skin involvement (2.7% vs. 25.6%, P = 0.002) were found in the MAP2K1 mutation group than in the BRAF V600E mutation group. There was no significant difference in other clinical features between the two groups (P > 0.05, Table 2).
No significant difference in clinical characteristics or laboratory examinations was found between the MAP2K1 mutation group and the no known mutation of the MAPK pathway group (P > 0.05, Table 2).

Analysis of treatment response and prognosis in the MAP2K1 mutation group
In the MAP2K1 mutation group, one patient, in whom only the left femur was involved, did not require chemotherapy according to the regimen and got improvement during the follow-up period, while the other 36 patients received firstline chemotherapy after diagnosis. Except for the patient who was not evaluated at the 6th week as planned, the ORR was 48.6% (17/35), and the DCR was 85.7% (30/35) after 6 weeks of first-line chemotherapy. In the same period, the ORR and DCR of the BRAF V600E mutation and the no known mutation of the MAPK pathway groups were 52.8% (65/123) and 84.6% (104/123) and 56.4% (31/55) and 94.5% (52/55), respectively. There was no significant difference in ORR and DCR after 6 weeks of first-line chemotherapy among the three groups (P > 0.05, Table 2).
We compared the prognosis of the three groups after firstline treatment and analyzed the prognosis of the patients who received second-line chemotherapy in the three groups. Although the 2-year PFS of the BRAF V600E mutation group was significantly lower than that of the no known mutation of MAPK pathway group (56.2% ± 4.8% vs. 70.1% ± 6.6%, χ 2 = 4.545, P = 0.033, log-rank test), there was no significant difference in 2-year PFS between the MAP2K1 mutation group and the other two groups after first-line treatment (P > 0.05, Table 2, Fig. 2A). In terms of the efficacy of second-line chemotherapy, there was no significant difference in 2-year PFS between the MAP2K1 mutation group and the BRAF V600E mutation group, but the PFS of both groups were significantly lower than that of the no known mutation of the MAPK pathway group (χ 2 = 6.531, P = 0.011; χ 2 = 7.409, P = 0.006; log-rank test, Table 2; Fig. 2B).

Clinical characteristics, treatment response and prognosis between different mutated domains of the MAP2K1 mutation group
The 37 patients with MAP2K1 mutations were divided into 2 groups: the mutation of the negative regulatory domain group (n = 30) and the mutation of the catalytic core domain group (n = 7). We compared the clinical and laboratory characteristics of the two groups and found  (1) p.Q56_V60del c.166_180del (2) p.Q56_V60del c.167_181del (3) p.Q56_G61delinsR c.168_182del (2) p.K57_G61del c.169_183del (1) p.K57_G61del c.170_184del (5) p.Q58_E62del c.172_186del (12) p.Q58_E62del Kinase domain c.294_311del (1) p.I99_K104del c.302_307del (4) p.E102_I103del c.304_309del (1) p.E102_I103del c.308 T > A; c.389A > G (1) p.I103N; p.Y130C that there was no significant difference in clinical features (including age, sex, and organs involved) or laboratory examinations (P > 0.05). In addition, no significant difference in 2-year PFS was found between the two groups after first-line treatment (61.7% ± 10.7% vs 85.7% ± 13.2%, χ 2 = 0.528, P = 0.468, log-rank test). A total of 7 patients in the negative regulatory domain group received second-line treatment, yet three of them had progression or relapse, and the 2-year PFS was 35.7% ± 26.7%. No patient in the catalytic core domain mutation group received second-line chemotherapy. However, there was no statistical difference in the rate of switching to second-line treatment between the two groups (23.3% vs. 0%, P = 0.306).

Analysis of prognostic factors in the MAP2K1 mutation group
We further explored the prognostic factors of MAP2K1mutated patients. Except for age at disease onset, which was grouped according to the median, other clinical features were grouped according to presence/absence, and laboratory examinations were grouped according to the normal reference range. Statistical differences in 2-year PFS after first-line treatment among different groups were compared. Univariate analysis showed that MS-LCH, lung involvement and elevated CRP were associated with poor prognosis in the MAP2K1 mutation group (P < 0.05, Table 3). The statistically significant factors above were included in the multivariate Cox proportional hazards model. After adjusting for confounding factors, lung involvement was an independent risk factor for poor prognosis in patients with MAP2K1-mutated LCH [Hazard ratio (95% CI) = 6.312 (1.769-22.526), P = 0.005] (Table 3). It was unusual that lung involvement was significantly associated with bony thorax involvement (5/17 vs. 0/20, P = 0.014; Table 4; Supplementary Figs. 1,2,3,4 and 5). In addition, more progression or relapses could be found in patients with bony thorax involvement (8/17 vs. 2/20, P = 0.023), yet involvements in other sites of bones, such as craniofacial bone involvement (8/26 vs. 2/11, P = 0.688) and limb bone involvement (5/12 vs. 5/25, P = 0.240), were not correlated to disease progression or relapse. Thus, the independent prognostic significance of lung involvement in MAP2K1 positive patients may be possibly due to the co-occurrence of bony thorax involvement.

Discussion
MAP2K1 kinase is an important component of the MAPK pathway, which is activated by upstream RAF kinase and further activates downstream ERK kinase to regulate the growth and proliferation of cells. MAP2K1 mutations occur in more than 1% of neoplastic diseases, with a higher mutation frequency in diseases such as histiocytic disorders, melanoma and hairy cell leukemia. Studies have shown that point mutations in MAP2K1 are predominant in melanoma and hairy cell leukemia (Williams et al. 2020), while deletion mutations are common in LCH (Nann et al. 2019;Chakraborty et al. 2014). The study presented here included the largest number of patients with MAP2K1-mutated LCH to date. The results showed that MAP2K1 mutations were concentrated at p.53-62 and p.98-103, located in the negative regulatory region and catalytic core domain of MAP2K1 kinase, respectively. Therefore, these mutations lead to abnormal activation of MAP2K1 kinase, which further activates ERK kinase and induces LCH (Alayed et al. 2016).
In this study, two point mutations in MAP2K1 were detected in one patient in the same biopsied tissue. Kamionek et al. (Kamionek et al. 2016) reported that two point mutations were also found in the same pulmonary nodule in a patient with LCH (p.A26T, p.S24N), but only one point mutation (p.A26T) was found in another pulmonary nodule in the same patient, which may indicate clonal evolution of independent nodules arising from a common progenitor cell. Because single-cell sequencing of the biopsied tissue of this patient was not performed and the mutations of other lesions were not detected, it is not clear whether the two mutations originated from one or two clones.
By comparing the clinical features of patients with MAP2K1 mutation and the BRAF V600E mutation, it was found that BRAF V600E -mutated LCH occurred earlier with a younger age of disease onset, and most of the patients  had RO (especially liver) and skin involvement, while the patients with MAP2K1 mutation mostly had SS-multiple bone involvement. These results are similar to those of previous studies (Cai et al. 2019).
The differences in the above clinical features are related to the functional characteristics of the MAP2K1 and BRAF mutation sites. The BRAF V600E mutation is a RAS-independent kinase activation mutation (class I BRAF mutation), causing high-level BRAF kinase activity that is not regulated by upstream signals and highly activates downstream ERK kinase (Yaeger et al. 2019). Mutations of amino acids 53-62 of MAP2K1 kinase are located in the negative regulatory region and are RAF-regulated mutations (class II MAP2K1 mutations). Thus, the activity of MAP2K1 mutated at these sites is regulated by upstream RAF kinase, and the phosphorylation level of ERK kinase is not as high as that which results from the BRAF V600E mutation. Mutations of amino acids 98-103 are located in the catalytic core of MAP2K1 kinase and are RAF-independent mutations (class III MAP2K1 mutations). MAP2K1 mutated at these sites is not regulated by upstream signals and highly activates downstream ERK kinase (Gao et al. 2018). Therefore, patients with the BRAF V600E mutation often have multiple system or RO involvement, a stronger inflammatory response and disease activity, such as high levels of CRP, IFN-γ, IL-10 and IL-6, and fever, all of which are related to the poor efficacy of chemotherapy (Kobayashi et al. 2018;Heritier et al. 2016). However, we did not find a difference in prognosis between the MAP2K1 mutation group and the BRAF V600E mutation group or the relationship between the two mutated domains of MAP2K1 kinase and the clinical features and prognosis. This is probably due to the influence of short follow-up time and small sample size, especially only seven patients carried mutations in the catalytic core domain. Thus, more samples with mutations in the catalytic core domain were needed to explore the effects of the two mutated domains on clinical characteristics and prognosis. In addition, BRAF kinase is located upstream of MAP2K1 kinase, and its substrate also includes MAP2K2 kinase, which may affect the clinical characteristics and prognosis and needs further study. However, it was noted that 7 out of 30 patients with mutations in negative regulatory region had progression during first-line chemotherapy and switched to second-line chemotherapy, moreover, 3 patients had progression or relapse. Although no statistical significance was found between patients with mutations in the two domains possibly due to small sample size, it still suggested the association of mutations in negative regulatory region with tolerance to chemotherapy. MEK inhibitors may be novel choices for these patients (Gao et al. 2018).
In this study, the clinical and laboratory characteristics, response rate after 6 weeks of first-line treatment and 2-year PFS of first-line treatment in the MAP2K1 mutation group were not significantly different from those in the no known mutation of the MAPK pathway group, which may be related to the greater heterogeneity of the latter. Those patients may carry other types of genetic aberrations which need to be identified. These aberrations may contribute to pathogenesis of LCH by activating MAPK pathway and affect the clinical and biological features of LCH. On the other hand, the observation that second-line chemotherapy resulted in a 2-year PFS of 100% in the patients without known mutations, which was significantly higher than that in the patients with BRAF V600E and MAP2K1 mutations, implies usefulness of second-line chemotherapy in treatment these patients. Thus, our findings suggest that further identification of new genetic mutations in LCH is of great significance for clinical stratification and precise treatment. In LCH, somatic and heterogenic mutations within the MAPK pathway affect myeloid progenitor and lead to the activation of kinases, which affects the recruitment of monocytes/macrophages and the differentiation of T cells by regulating the activities of many transcription factors and enzymatic proteins and can ultimately lead to the formation of pulmonary lesions (Radzikowska et al. 2017). There is a clear correlation between lung involvement and tobacco exposure in adolescents and adults with LCH, and more than 2/3 of these patients have respiratory symptoms, such as cough and dyspnea (Rodriguez-Galindo et al. 2020). However, only 10% of children with lung involvement have respiratory symptoms, which are diagnosed mainly by high-resolution computed tomography (Wang et al. 2018). This lack of symptoms may delay diagnosis in the absence of routine radiological monitoring. In this study, the five MAP2K1-mutated LCH patients with lung involvement had no history of tobacco exposure, and only one of them had a mild cough. The rest were diagnosed by high-resolution computed tomography, manifesting as scattered/bilateral cystic changes or reticulonodular changes. Interestingly, in this study, lung involvement is an independent risk factor for poor prognosis in patients with MAP2K1 mutations. As lung involvement may occur mostly in patients with bony thorax involvement and more progression or relapses could be found in patients with bony thorax involvement, the involvement of bony thorax may contribute to the poor prognosis of patients with lung involvement, the underlying mechanisms of which remaining to be further studied. In addition, the sample size of this study is small and does not meet the requirements of the events per variable, so the results may not be robust enough, and the reliability needs to be confirmed by further research. In this study, the efficacy of second-line chemotherapy with cytarabine as the main drug in the MAP2K1 mutation group was significantly worse than that in the no known mutation of the MAPK pathway group. Three of seven patients with MAP2K1-mutated LCH had progression or relapse during the third course of second-line chemotherapy, 3 months after drug withdrawal, or maintenance chemotherapy. This suggests the application value of MEK-targeted inhibitors such as trametinib in patients with MAP2K1 mutations, especially in patients with poor response to conventional chemotherapy (Baiocchi et al. 2014). Lorillon et al. (Lorillon et al. 2018) reported a patient with MAP2K1mutated LCH (p.E102_I103del) with lung involvement. The pulmonary lesion progressed consistently during the treatment with cladribine and improved after treatment with trametinib with good tolerance. Papapanagiotou et al. (Papapanagiotou et al. 2017) reported a pituitary-involved patient with MAP2K1-mutated LCH (p.E102_I103delinsEI). During treatment with trametinib, the dosage of desmopressin to control diabetes insipidus was reduced, and the relapsed lesion could be quickly reduced by the reuse of trametinib. However, it has been found that p.Q56_G61de-linsR, p.C121S/G128D, and p.98_104delinsQ mutations (of which only the first was found in this study) may be related to resistance to trametinib (Nelson et al. 2015;Azorsa et al. 2018), so the efficacy and safety of targeted therapy need to be further studied.
In conclusion, MAP2K1 mutations are concentrated at amino acids 53-62 and 98-103, and c.172_186del is the most common mutation site in pediatric LCH. Compared with patients with the BRAF V600E mutation, patients with the MAP2K1 mutation were mainly characterized by more SSmultiple bone involvement, less RO and skin involvement and later disease onset. The prognosis of patients with lung involvement is poor and should be given more attention in future studies.