Abstract
从肺Langerhans的患者的Langerhans样细胞中检测BRAF和NRAS的细胞中的BRAF和NRA。http://ow.ly/ccdg30f0tct
To the Editor:
肺Langerhans的细胞组织细胞增多症(PLCH)呈现为Langerhans的细胞的积累和肺部表达的其他表达树枝状细胞(LCH细胞),引起双侧结节和空腔,这些结节通常限制在成年吸烟者的上肺肺部[1–3]。The PLCH lung nodules appear to be the origin of the cavities, which could also have a cyst-like appearance that gives rise to a differential diagnosis that includes both cavitary and cystic lung diseases [3]。A characteristic histopathologic feature of PLCH is destruction of the wall of distal airways by infiltration of LCH cells [3]。鉴于细胞病因,已经提出LCH,特别是PLCH可能代表肿瘤或反应性条件[4,5]。Currently, the diagnostic criteria of LCH include the demonstration of CD1a- and CD207-positive LCH cells in the LCH lesions [6]。作为脂质呈递分子,CD1a在表达兰格蛋白的细胞上大量表达并积聚在Birbeck颗粒中,在此中,它与langerin共归因于[6]。TheBRAF在两项独立研究中,在38%至64%的LCH病变中检测到LCH中的突变,包括PLCH病例[7,8]。最近,m我们的et al。[9]提出了一个了不起的发现NRAS, in addition toBRAFmutations, occur in PLCH lesions. The findings of an abnormal cell and an oncogenic mutation are consistent with the proposal that LCH is a neoplastic disease.
We hypothesised that if Langerhans’ cells were indeed neoplastic, and thus, similar to other cancers, circulating tumour cells and DNA could be isolated and might aid in diagnosis and treatment. Circulating tumour cells accompany tumour invasion of the bloodstream. Circulating cell-free tumour DNA (cfDNA) containing oncogenetic mutations is under investigation as a specific biomarker for the diagnosis and monitoring of patients with different cancer types [10]。Expanding knowledge of molecular abnormalities that drive human cancers offers the promise of personalised therapies focused on particular genetic lesions. To date, there is no evidence showingBRAF和NRAS循环的Langerhans样CD1A中的突变+cells and DNA of PLCH patients. Here, we used anti-CD1a antibody to identify circulating Langerhans-like cells by fluorescence-activated cell sorting (FACS). In addition, we evaluated mutations in circulating LCH cells in peripheral blood as well as plasma, as biomarkers in PLCH.
The conditions PLCH and lymphangioleiomyomatosis (LAM) are two rare neoplastic lung diseases of unknown aetiology and different pathogeneses [4,11]。As a slowly progressive multisystemic disease, LAM primarily affects women of reproductive age, and is characterised by cystic lung destruction. Small clusters of smooth muscle-like cells (LAM cells) can be found in the walls of the cysts and in the pulmonary interstitium [11]。这些LAM细胞是该疾病独有的,并且在肿瘤抑制基因中包含突变,结核性硬化症复合物1(TSC1)或者TSC2[12]。Circulating LAM cells, which have been isolated based on cell surface markers and cell density [13], have been demonstrated in patients with sporadic LAM and those with TSC [14]。Detection of circulating cells is significantly reduced in patients treated with sirolimus (rapamycin), an inhibitor of the mechanistic target of rapamycin (mTOR).
Patients who were diagnosed with PLCH or LAM were seen at the National Institutes of Health Clinical Research Center in protocols (95-H-0186, 96-H-0100) approved by the National Heart, Lung, and Blood Research Institutional Review Board. The diagnosis of PLCH was based on clinical, radiographic, lung biopsy and histopathologic criteria (figure 1a, b). Cells from blood were sorted on the basis of cell surface markers that have been shown to identify LAM cells (CD235a, CD45) or Langerhans-like CD1a+cells (CD1a) [6,13]。外显子15中的突变BRAFgene and exon 3 of theNRAS通过基于聚合酶链反应(PCR)的直接测序分析基因,使用每个细胞分数分离的DNA进行直接测序。鉴定的基于单元和PCR的方法BRAF和NRASmutations in circulating cells.
当它发生在内部器官,华尔街可能脸上t from mutations in stem cells in bone marrow or circulating cells [15]。M伊尔恩et al.[16] reported a developmental pathway leading to the formation of human Langerhans’ cells from circulating CD14-和CD1C+dendritic cell precursors in peripheral blood. In other studies, Berreset al.[17] found that patients with multisystemic LCH carried theBRAFmutation in circulating CD11c+/CD14+cell fractions as well as in bone marrow CD34+progenitor cells. Kobayashi和Tojo[18]评估BRAF来自八名LCH患者的CFDNA突变,仅报告了三名多系统的LCH患者测试阳性BRAF-V600E。在这里,我们证明了BRAF-V600E后期隔离的PLCH患者的肺部病变突变限制为肺部。此外,在Oncoquick(Greiner Bio-One GmbH,Frickenhausen,Germany)中循环的LCH细胞中发现了相同的突变。+cell fraction. The mutation was not found in the circulating LAM cell population (CD45-/CD235+/-)(figure 1c). Moreover, we detected theNRASCD1A中同一PLCH患者的循环细胞中的突变+cell fraction (figure 1d),但不在Oncoquick(Greiner Bio-One GmbH)密度梯度增强的细胞派系或PLCH肺病变中。这一发现的原因可能是M我们的et al。[9]指出,那BRAF和NRASmutations could be observed from different clones of lung lesions. Moreover, the allele frequency of theNRASmutation was often low. TheBRAF和NRAS在CFDNA中未发现突变,这表明在PLCH中,循环细胞可能比CFDNA具有更高的敏感性,以检测这两者BRAF和NRASmutations. Furthermore,BRAF和NRASmutations were not identified in circulating cells from two LAM patients, indicating that these two point mutations could be considered as evidence, consistent with a PLCH diagnosis.
孤立的PLCH很少见,其发病机理的定义很差。但是,香烟被视为成年人疾病进展的危险因素[1]。Recurrence of PLCH after double lung transplantation is consistent with a non-pulmonary source of LCH cells [19]。我们的结果也表明BRAF和NRAS突变可能来自不同的来源。
TheBRAFgene is a member of the皇家空军family of serine threonine kinases that contributes toRAS-RAF-MAPK信号[20,21]。Somatic-activatingBRAFgene mutations are observed in a range of cancers, including melanoma, colorectal and lung cancer [21]。之间BRAF突变,V600E是迄今为止最常见的,它导致组成性活性蛋白。已经提出了不减弱的激活我K-ERK途径导致细胞增殖,生存和最终恶性进展[20]。Here, we confirmed that LCH-derivedBRAFmutations could be enriched in CD1a+fractions of circulating cells, consistent with the classification of PLCH as a neoplastic disorder.
In addition, we also detected NRAS, as might be expected based on the study by M我们的et al。[9]。TheNRAS基因包括拉family由三个成员组成的Oncogenes:HRAS, KRAS和NRAS[22]。Each isoform of拉displays preferential coupling to particular cancer types. TheNRAS突变存在于25–30%的黑色素瘤[23], in 0.7% of patients with lung cancer [24], and in 10.3% of patients with myeloid leukaemia [25]。
In summary, we report a procedure for the detection ofBRAF和NRAS循环的Langerhans样CD1A中的突变+细胞。该程序可能有助于将PLCH与其他空洞和囊性肺疾病区分开。
Acknowledgements
We thank J. Philip McCoy and Leigh Samsel (both Flow Cytometry Core Facility, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA) for help with the FACS analysis.
脚注
Support statement: The study was supported by the Intramural Research Program, National Institutes of Health, National Heart, Lung, and Blood Institute.
Conflict of interest: None declared.
- 已收到March 13, 2017.
- AcceptedJuly 11, 2017.
- 版权所有©ERS 2017