3.1 Characteristics of MAP2K1 gene mutation
In this study, thirteen MAP2K1 mutations were detected in 37 patients with LCH (Table 1), accounting for 11.1% (37/334) of all patients. The most common mutation was c.172_186del (p.Q58_E62del), accounting for approximately 32.4% (12/37); the nucleotide mutations of c.165_179del (p.Q56_V60del), c.166_180del (p.Q56_V60del), c.294_311del (p.I99_K104del), and c.389A>G (p.Y130C) have not been reported in the literature, but the amino acid mutation of p.Q56_V60del caused by the first two nucleotide mutations has been reported (Hayase et al. 2020).
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.
3.2 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 BRAFV600E 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 BRAFV600E 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 BRAFV600E mutation group (χ2=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 BRAFV600E mutation group (χ2=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 BRAFV600E mutation group. There was no significant difference in other clinical features between the two groups (P>0.05, Table 2).
Compared with the BRAFV600E mutation group, the levels of C-reactive protein (CRP), IFN-γ, IL-10 and IL-6 in the MAP2K1 mutation group were significantly lower (P<0.05). There was no significant difference in other laboratory examinations (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).
3.3 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 first-line 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 BRAFV600E 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 first-line treatment and analyzed the prognosis of the patients who received second-line chemotherapy in the three groups. Although the 2-year PFS of the BRAFV600E 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%, χ2=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. 1A). 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 BRAFV600E mutation group, but the PFS of both groups were significantly lower than that of the no known mutation of the MAPK pathway group (χ2=6.531, P=0.011; χ2=7.409, P=0.006; log-rank test, Table 2, Fig. 1B).
3.4 Clinical characteristics, treatment response and prognosis between different mutated domains of the MAP2K1 mutation group
The 37 patients with MAP2K1 mutations were divided into two 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 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%, χ2=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).
3.5 Analysis of prognostic factors in the MAP2K1 mutation group
We further explored the prognostic factors of MAP2K1-mutated 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 [HR (95% CI) = 6.312 (1.769-22.526), P = 0.005] (Table 3). It was noteworthy that lung involvement was significantly associated with bony thorax involvement (5/17 vs. 0/20, P=0.014, Table 4). 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.