Background
Lymphatic anomalies (LAs), including generalized lymphatic anomaly (GLA) and kaposiform lymphangiomatosis (KLA), are extremely rare diseases with severe symptoms and poor prognosis [
1]. KLA is categorized as a novel subtype of GLA in the International Society for the Study of Vascular Anomalies (ISSVA) classification updated in 2018 [
2]. It is described as an aggressive disease of the lymphatic system and has foci of “kaposiform” abnormal spindle lymphatic endothelial cells; however, the pathogenesis of the patients remains unknown [
3].
Recently, genetic research has attempted to elucidate the actual conditions and pathogenesis of vascular anomalies [
4]. Low-level somatic mutations of PI-3 kinase or the RAS pathway have been detected in samples from vascular anomalies [
4]. In recent studies, the
NRAS c.182A > G (p.Q61R) mutation was detected in affected lesions of KLA and GLA patients [
5,
6]. However, a tissue biopsy can lead to severe complications, such as bleeding or lymphatic leakage. Therefore, development of noninvasive diagnostic methods may not only provide a more accurate diagnosis, but also decrease the risk of complications. Analysis of cell-free DNA (cfDNA), so-called liquid biopsy, is becoming a promising clinical application for molecular testing and cancer detection [
7].
Here, we describe a case of KLA, in which a somatic mutation in the NRAS gene was detected both in a biopsy specimen and in cfDNA. Additionally, the same mutation was detected in cfDNA from the four other patients with KLA and from a tissue sample obtained from one of them. Our data therefore suggest that cfDNA analysis might be a useful method for diagnosing KLA.
Discussion
We here present the first study of liquid biopsy for NRAS mutation in KLA cases. In a first case with intractable KLA treated with sirolimus, which induced improvement of symptoms and tumor regression, WES of DNA samples from leukocytes and tumor tissues showed the presence of NRAS c.182A > G (p.Q61R) in 5% of alleles in tumor tissues, which was confirmed by TA cloning of the PCR product followed by sequencing. The NRAS mutation was also detected in cfDNA isolated from plasma and pleural effusion. Furthermore, we conducted target-specific NRAS mutation analysis by using cfDNA isolated from liquid sample of 7 patients (4KLA and 3GLA) and detected the targeted gene mutation in 80% (4/5) of KLA patients (P1, P2, P3, and P4). The genotyping of FFPE tissue showed that the same mutation was also detected in an affected lesion from a KLA patient (P2). These results suggested that this somatic oncogenic NRAS mutation may be involved in the pathogenesis of KLA, and cfDNA might be useful for diagnosing KLA.
KLA is recognized as a new entity that has an aggressive course and poor prognosis. Recent research has attempted to elucidate the actual conditions and pathogenesis of these diseases. KLA was classified by the World Health Organization as a rare tumor of lymphatic vessel origin with the capacity to metastasize [
10]. Although the ISSVA classification of 2014 categorized this condition as a provisionally unclassified vascular anomaly, KLA was categorized as a subtype of GLA in the ISSVA classification updated in 2018 [
2]. The key signals of KLA are thrombocytopenia, coagulation disorder, and hemorrhagic pericardial and pleural effusion or ascites. However, the pathogenesis and etiology of KLA are still unknown. GLA also involves the diffuse or multicentric proliferation of lymphatic vessels in several organ systems, and an appropriate diagnosis is difficult because the clinical findings of KLA and GLA overlap [
1]. Dilated malformed lymphatic channels lined by a single layer of endothelial cells are common to both GLA and KLA; the latter also has foci of patternless clusters of intra- or peri-lymphatic spindle cells associated with platelet microthrombi, extravasated red blood cells, hemosiderin, and some degree of fibrosis. Recently, two reports on cytokine analysis in patients with lymphatic anomalies showed that angiopoietin-2 and some cytokines are important markers for KLA [
11,
12]. However, differentiation of these diseases is challenging based on their phenotypic presentation alone, so further study is needed.
Although genetic analysis revealed somatic mutations in genes associated with the phosphoinositide 3-kinase (PI3K) pathway in patients with LAs [
4], little has been reported on the associated genetic abnormalities of KLA. In a recent study, Manevitz-Mendelson et al. reported the possibility that somatic
NRAS mutation causes GLA [
6]. Activating mutations in
RAS proto-oncogenes (
KRAS,
HRAS, and
NRAS) have been found in a variety of human malignancies, suggesting a dominant role in carcinogenesis [
13‐
15]. The authors isolated lymphangiomatosis endothelial cells from a GLA patient using CD31-coated magnetic beads and identified a somatic activating mutation in
NRAS in fewer than 30% of the alleles of the endothelial cells. The results of the study showed that the
NRAS mutation plays key roles in the regulation of angiogenesis and lymphangiogenesis. Barclay et al. reported that
NRAS mutations were detected in the affected lesions of KLA patients [
5]. We also found that KLA patients possessed the same
NRAS mutation. Although why the same
NRAS mutation could be associated with both GLA and KLA is not clear, it could be speculated that the confusion is due to not only the biology of these diseases but also the difficulty of the diagnosis. The pathogenesis of these diseases requires further investigation.
In a wide spectrum of RAS-related disorders, recently called RASopathies [
16], somatic
RAS mutations, especially somatic
NRAS mutation at codon 61 (p.Q61R/K), have been identified in a variety of human malignancies [
15]. Somatic
NRAS c.182A > G (p.Q61R) mutations have also been identified in nonmalignant cancers, including pyogenic granuloma [
17] and Langerhans cell histiocytosis [
18]. In contrast, germline
NRAS mutations have been identified in patients with Noonan syndrome, which is characterized by short stature, congenital heart disease, lymphatic abnormalities, chest deformity, and predisposition to malignant tumors [
16]. However,
NRAS p.Q61R mutation has never been identified in Noonan syndrome patients, suggesting that patients with germline p.Q61R mutation do not survive to birth because of its strong activation of the downstream pathway. In mosaic RASopathies,
NRAS p.Q61R mutations have been identified in a patient with Schimmelpenning syndrome [
19], patients with neurocutaneous melanosis [
20], and patients with cutaneous–skeletal hypophosphatemia syndrome [
21]. These results suggest that somatic or mosaic
NRAS p.Q61R mutations cause a broad spectrum of disorders, which depend on the lineages or cells in which they occur.
Low-frequency
NRAS mutation was identified in a chest mass in our patient. However, we were not able to identify in which cells this mutation arose.
NRAS mutations play a critical role in angiogenesis and lymphangiogenesis [
6]. We also performed ddPCR assays of the
NRAS p.Q61R mutation for other KLA and GLA patients who had not undergone WES (genetic) analysis because we did not have fresh frozen samples of their affected lesions. A total of 80% (4/5) of patients with KLA and 33.3% (1/3) of patients with GLA showed low-frequency
NRAS mutation in cfDNA isolated from plasma or pleural effusion. Additionally, the same mutation was detected in the FFPE tissue of another KLA patient (P2). Although the results of P3, 4, and 6 could be false positives because the minimum Poisson fractional abundance in their samples was statistically 0.0, the
NRAS p.Q61R mutation was apparently detected in tissue samples from two KLA patients (P1 and P2). Our results indicate the possibility of developing a novel diagnostic method, a so-called liquid biopsy, for KLA without the need for an invasive procedure.
The finding that
RAS mutation drives vascular anomalies including GLA and KLA provides potential opportunities to develop targeted therapies for current drug-resistant lesions. Treatment with an mTOR inhibitor, sirolimus, and an MEK inhibitor, trametinib, had an effect of reducing the viability of the affected cells through inhibition of the phosphorylation of AKT and extracellular signal-regulated kinase (ERK) [
6].
NRAS inhibition by these drugs might be a promising option for the treatment of these lymphatic anomalies. In our patient, sirolimus was effective to improve clinical symptoms and subcutaneous lesions. These genes are associated with the pathogenic etiology of lymphatic diseases and their inhibition might thus be a target for treatment.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.