Association between clinicopathologic characteristics and BRAFV600E expression in Chinese patients with Langerhans cell histiocytosis

Background The identification of V‐raf murine sarcoma viral oncogene homolog B1 (BRAF)V600E mutations has been recommended in patients with Langerhans cell histiocytosis (LCH) with difficult diagnosis and failure of first‐line treatment. The reported frequencies of BRAFV600E mutations vary in Chinese patients with LCH. Methods We conducted a retrospective analysis of LCH patients with a definitive pathological diagnosis who were hospitalized between 2013 and 2017. The BRAFV600E mutations were detected with the human BRAFV600E amplification refractory mutation system‐PCR (ARMS‐PCR) kit from the collected tissue samples. Results This study consisted of 46 male (68.7%) and 21 female (31.3%) patients, with a mean age of 29.1 years (range, 2–76 years). Most were adults (45/67.2%) with the multisysytem‐LCH (MS‐LCH) disease subtype (49/61.3%). The overall frequency of BRAFV600E mutations was 22.4% (15 of 67 patients), confirmed by PCR analysis. These mutations were not closely correlated with age (nonadults vs. adults = 5/22.7% vs. 10/22.2%, P = 0.54), gender (female vs. male = 9/19.6% vs. 6/28.6%, P = 0.61), LCH classification type (single system: MS‐risk organ+: MS‐risk organ− = 3/16.7%: 12:28.6%: 0, P = 0.19) or prognosis (cured: improved/stable: exacerbated: died = 4/44.4%: 19.2%: 20%: 0, P = 0.37). There were 33 patients (49.2%) with lung involvement, and 12 patients (36.3%) underwent lung biopsies; after screening, four patients were diagnosed with solitary pulmonary LCH, all of whom were negative for BRAFV600E mutations. Conclusion The BRAFV600E mutation rate in patients with LCH was lower than those reported in other studies. In addition, BRAFV600E mutations might not be correlated with age, gender, LCH classification type or prognosis for Chinese cases.


What this study adds
The BRAF V600E mutation rate in LCH varies.

Introduction
As V-rf murine sarcoma viral oncogene homolog B1 (BRAF) V600E mutations are present in approximately half the samples from patients with Langerhans cell histiocytosis (LCH), and treatment with BRAF inhibitors has been reported to be effective for some patients with LCH, the identification of BRAF V600E mutations is recommended for all patients with LCH with difficult diagnosis and failure of first-line treatment (grade C2). 1 The reported frequencies of BRAF V600E mutations in patients with LCH vary among different ethnicities or countries. 2 No BRAF V600E mutations were reported in adults with LCH in the study by Tong et al., 3  for Chinese adult patients with LCH and 46.4% and 32% for all recruited patients with LCH, respectively. Here, we retrospectively analyzed BRAF V600E mutations and the clinical features of patients with LCH with a positive pathological diagnosis at our hospital over the last five years.

Methods Patients
A computer-assisted search for patients hospitalized at Peking Union Medical College Hospital from January 2013 to December 2017 identified 167 patients diagnosed with LCH according to the 2016 World Health Organization criteria. 1 Most patients were diagnosed by our pathologist in consultation with biopsy/surgical samples from other hospitals. Finally, 67 patients with complete medical records, radiologic images and pathological specimens were retrospectively recruited into this study. All patients were followed-up every one to six months, depending on disease activity and treatment. The mean follow-up period was 36.8 months, ranging from seven to 59 months.
All patients underwent chest CT scans, cerebral magnetic resonance imaging, bone scintigraphy scans, whole body bone plain films, and bone marrow biopsies, and 26 patients underwent 18F fluorodeoxyglucose (FDG) positron emission tomography (PET) scans. The involved sites were defined according to the typical imaging scans and/or the pathological manifestations.
The following information was analyzed: age, sex, clinical manifestations, serological results, radiologic findings, pathological manifestations, treatments and outcomes. Hematoxylin-eosin (HE) staining and immunohistochemical (IHC) staining analysis of CD68, CD1a, CD207, and S100 were performed for all enrolled patients. All glass slides were reviewed and scored by two pathologists (R.E.F. and J.L.), who were blinded to the molecular results. The two pathologists independently came to a consensus diagnosis based on the WHO recommendations for all enrolled patients. 6 All patients and/or their relatives provided written informed consent. This study was approved by the ethics committee of Peking Union Medical College Hospital (JS-1127, ZS-1058), in accordance with the Declaration of Helsinki.

DNA extraction
Tumor DNA was extracted from formalin-fixed, paraffinembedded tissues from our pathological sample bank. Following HE and immunohistochemical staining, the samples with the highest CD1a-positive histiocyte density were selected for further BRAF mutation analysis.
DNA was extracted from the collected tissue samples using the QIAGEN QIAamp DNA FFPE Tissue Kit (154051332, QIAGEN China (Shanghai) Co. Ltd., Shanghai, China), following the manufacturer's protocol.

Detection of BRAF V600E mutations by PCR analysis
The BRAF V600E mutations were detected with the human BRAF V600E ARMS-PCR kit (P216010801Y, Amoy Diagnostics Co. Ltd., Xiamen, China), which has been approved by the China Food and Drug Administration. The extracted DNA quality was evaluated by amplification of a housekeeping gene following the instructions in the HEX channel. PCR was performed on the PCR System 7500 (ABI) system for 47 cycles according to the instructions supplied with the BRAF V600E ARMS-PCR kit. Both negative and positive controls were included in each set of amplifications. The sequencing results were analyzed and interpreted according to the manufacturer's protocol.

Statistical analysis
Data were analyzed using the Statistical Analysis System (SAS) version 9.0 software package. Quantitative variables are presented as the mean AE standard deviation (SD), and categorical data are presented as the frequency and percentage in the text and figures.

General clinical characteristics
The general clinical characteristics of the 67 enrolled patients are summarized in Table 1 Table 3. Lung involvement was more common in patients with the MS-LCH subtype (P = 0.006), and patients with thyroid gland involvement (100% vs. 36.2%, P = 0.0073). After either surgical or fine needle biopsy, eight patients were diagnosed with LCH with thyroid involvement. The imaging studies of these eight patients showed classic lung shadows indicating diffuse cysts with bizarre shapes. In our cohort, most of the patients with LCH with lung involvement were patients with systemic LCH. All four patients with PLCH were smokers. Three were cured after quitting smoking and avoiding secondhand smoke for four to six months, without medications. The fourth patient with PLCH was diagnosed three months prior to this manuscript being written, and his lung infiltrations improved after quitting smoking, without taking medication. Although all patients with PLCH had good prognoses, there was no difference in prognosis between patients with LCH with and without lung involvement.

Discussion
The BRAF gene is located on chromosome 7q34, and is a member of the RAF kinase family. Although mutations of BRAF have been identified in a large number of solid tumors, it was first reported by Badalian-Very et al. in 2010 that BRAF V600E expression was identified in 57% of samples from patients with LCH. 8 Following this study, there were several studies that focused on the mitogenactivated protein kinase (MAPK) pathway signal transmission, including BRAF V600E , 2,8-13 mitogen-activated protein kinase 1 (MAP2K1) and NRAS. [14][15][16][17] It was reported that the activating BRAF V600E mutation rate ranged from 35% to 60% in different studies using PCR or immunohistochemistry (IHC) staining 2,[8][9][10][11][12] in patients with LCH. According to the review by Selway et al. the BRAF V600E mutation rate was 51.13% in 397 patients with LCH. 18 The mutation rate varies across different studies and different races. The BRAF V600E mutation rates reported in Chinese patients with LCH were 0 in the study by Tong et al. 3 56% in the study by Wei et al. and 22.5% in our study. 4 The patient age distribution, involved sites, stage, and different detection tests might influence the BRAF V600E subtype. 3,8,19 Badalian-Very et al. 8 13,18 the rate of the BRAF V600E mutation was increased in patients who experienced involvement of higher risk organs, such the liver and spleen. In the study by Selway et al., 18 although 75% of the biopsied liver samples were positive for BRAF V600E mutations, none of our patients with LCH with liver involvement had BRAF V600E mutations.
The lung is a commonly involved site in patients with LCH, and PLCH has been commonly reported in previous studies. However, only four patients (6%) in our cohort were diagnosed with solitary PLCH. Most of the previous studies did not show the completed screening tests for the enrolled patients with LCH. 2,4,8,[10][11][12][13][14][15][16] In our study, all 67 patients underwent strict screening tests including chest CTs, cranial MRIs, bone scans and bone marrow biopsies, with the exception of detailed serum tests. In addition, 26 patients underwent 18F FDG-PET-CT scans, which is a useful and sensitive tool for the identification of active lesions, the stratification of disease stages, and the monitoring of a therapeutic response in patients with LCH. 20 Most of our patients with lung involvement were diagnosed with the MS-LCH subtype after these screening tests, and only four patients were diagnosed with solitary PLCH.
Solitary PLCH is a smoking-related non-neoplastic disease, and the study by Yousem et al. failed to show clonality in patients with PLCH. 21 However, some studies reported BRAF V600E mutation rates ranging from 28% to 89% in patients with PLCH. 22 In our study, all four PLCH patients smoked, and all were negative for BRAF V600E mutations. Three of them were cured, and the fourth patient experienced improvement after quitting smoking and avoiding secondhand smoke, without medications. The natural history of PLCH varied widely. Some patients with PLCH may remit or stabilize after quitting smoking. However, others may develop pulmonary fibrosis, pulmonary hypertension, and respiratory failure, even after chemotherapy. There has been no further analysis of the BRAF V600E mutation status for those self-cured PLCH patients in most studies.
There were several limitations in our study. First, all enrolled patients had a definitive diagnosis of LCH and had complete clinical records, radiological images and pathological specimen, which could cause a selection bias. The BRAF V600E mutations in our study were detected by PCR analysis, and the DNA was extracted from formalin-fixed paraffin-embedded tissues, which could minimize the sensitivity. Second, not all enrolled patients underwent lung biopsies. There were 33 patients (49.2%) who had lung involvement, but only 12 patients (36.3%) underwent lung biopsies. LCH was diagnosed with extrapulmonary tissue biopsies, even for some cases with diffuse pulmonary infiltrations. However, for these cases, the clinical characters and extrapulmonary pathological manifestations were sufficient for the diagnosis of LCH, and to avoid excessive damage, lung biopsies were not performed. Third, peripheral blood BRAF V600E mutations for LCH cases had been previously reported, and the mutation status might be associated with the disease burden and therapy response. [23][24][25][26] As our study was retrospective, we were unable to obtain samples from all enrolled cases, and most of them were treated when we connect with them. Peripheral blood BRAF V600E mutation status will be analyzed in our future prospective studies, especially for MS-LCH cases.
In conclusion, the BRAF V600E mutation rate in patients with LCH was lower than in some reported studies. In addition, BRAF V600E mutations might not correlated with age, gender, LCH classification type, or prognosis in our patients with LCH.