Response to mitogen‐activated protein kinase inhibition of neurodegeneration in Langerhans cell histiocytosis monitored by cerebrospinal fluid neurofilament light as a biomarker: a pilot study

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia with highly variable clinical presentation. ¹ Granulomatous lesions of bone, skin, and lungs (particularly in adults) are most common, but the liver, spleen, bone marrow, and central nervous system (CNS) may also be affected. ¹ CNS involvement (CNS LCH) often causes endocrinopathies, most commonly diabetes insipidus (DI), but may also cause a debilitating slowly‐progressive neurodegeneration. ¹ , ² Notably, a population‐based study reported that at least 24% of all children with LCH develop signs of neurodegenerative CNS LCH (ND‐CNS‐LCH) on magnetic resonance imaging (MRI). ³ Thus, a strategy for early detection, treatment, and monitoring of ND‐CNS‐LCH is imperative.

In the current international treatment protocol (LCH‐IV), one‐year monotherapy with low‐dose cytarabine or intravenous immunoglobulin is suggested for patients with clinically manifest ND‐CNS‐LCH. ⁴ , ⁵ However, these treatment attempts have only had limited effects. Importantly, LCH has been associated with oncogenic somatic mutations, predominantly in BRAF and MAP2K1, resulting in constitutive activation of the mitogen‐activated protein kinase (MAPK) pathway in LCH lesions. ⁶ This has led to successful treatment with targeted MAPK pathway inhibition in LCH. ⁷ , ⁸ However, the evidence for therapeutic efficacy of MAPK inhibition (MAPKi) in established ND‐CNS‐LCH is limited. ⁹

Neurofilament light‐chain protein (NFL) in the cerebrospinal fluid (CSF) is a sensitive and well‐established biomarker of neuroaxonal damage, irrespective of cause or clinical diagnosis. ¹⁰ We have previously reported on elevated CSFNFL levels and monitoring in ND‐CNS‐LCH. ¹¹ , ¹²

In our endeavour to reduce progressive neurodegeneration in LCH, we initiated treatment with MAPKi, between January 1, 2020, and June 30, 2020, in five children affected by CNS LCH. In parallel we monitored NFL and other biomarkers [tau, phospho‐tau, and glial fibrillary acidic protein (GFAP)] in CSF. Clinical, laboratory and neuroradiological findings as well as treatments and outcome are presented in Table I, ¹³ and more detailed clinical information in the Supplementary Material. Four patients, aged 2–17 years, had further developed ND‐CNS‐LCH with clinical and neuroradiological abnormalities; all had cognitive difficulties and two had additional neurological symptoms. Prior to treatment with MAPKi, three children had each received at least seven different LCH‐directed drugs each (Table I), with no or limited clinical, neuroradiological, or CSF NFL regression.

Table 1.

Clinical and laboratory findings in five children with CNS LCH.

	Patient 1	Patient 2	Patient 3	Patient 4	Patient 5
LCH prior to CNS LCH
Sex	Male	Male	Male	Male	Female
Age at LCH diagnosis	6 mo	32 mo	3·5 year	15 mo	23 mo
Craniofacial bones involved at diagnosis	Orbita, temporal, sphenoid	Temporal	None (parental report)	Temporal, sphenoid, orbita, maxilla, zygomaticusa	Orbita
All organs involved since diagnosis (prior to MAPKi)	Bone, CNS, probably skin	Bone, skin, CNS, possibly lungs	LN, lungs, CNS	Bone, skin, LN, liver, spleen, CNS, bone marrowa,b	Bone, skin, CNS, bone marrowb
Maximal extent of disease	MS RO−	MS RO−	MS RO−	MS RO+	MS RO−
Disease Activity Score at maximal extentc	1	1	N/A	7	3
Therapy before CNS LCH diagnosis	VBL, Pred, VCR, Ara‐C, MTX, 6‐MP, Dexa	VBL, Pred	VBL, Pred, VCR, Ara‐C, MTX	VBL, Pred, MTX, 6‐MP, HD‐MTX, CsA, 2‐CdA	VBL, Pred
Treatment effect	After reactivations finally NAD in bone but ND‐LCH	Bone: AD better; CNS: AD worse (DI)	AD better	After multiple reactivations, finally NAD except ND‐LCH	Skin and bone: NAD; CNS (pituitary stalk): AD better.
CNS LCH prior to MAPK inhibition
Age at CNS LCH diagnosis	14 mo	37 mo	7 year	13 year	23 mo
Endocrinopathies at CNS LCH diagnosis	DI	DI	DI, hypothyroidism. Later also GH deficiency	None	DI. Later also GH deficiency.
Cognitive affection	Yes	No	Yes	Yes	Yes
Neurological symptoms	Balance problems	None	None	None	Subtle leg weakness
Disease Activity Score prior to MAPKic	0	0	1	0	1
Elevated sedimentation rate (mm/h)	24	6	19	2	7
CNS LCH MRI findings prior to MAPKi	Absent ‘bright spot’d, normal pituitary stalke Increased T2/FLAIR signal in dentate nuclei	Thickened pituitary stalk, absent ‘bright spot’d Enlarged bilocular pineal cysts (present at diagnosis). No known NDg	Enlarged pons, adjacent medulla oblongata and mesencephalon with diffusely increased signal. Increased T2/FLAIR signal in globi pallidi and amygdala. Thickened pituitary stalkf. Partial improvement after 2‐CdA.	Increased T2 signal in dentate nuclei and thalamus.	Thickened pituitary stalk, absent ‘bright spot’d Increased T2/FLAIR signal in dentate nuclei. Reduced white‐matter volume. Multiloculated pineal cysts.
Treatments given for CNS LCH prior to MAPKi	Ara‐C	None	Pred, VCR, Ara‐C, 2‐CdA, MTX, 6‐MP	None	None
Treatment effect; NFL before‐after therapy	MRI unchanged; NFL 890‐710 ng/l	–	CNS initially AD better/ stable, then AD worse; NFL 1210‐810 ng/l	–	–
CNS LCH on MAPK inhibition
Age at start of MAPKi	6·5 year	39 mo	12·5 year	18 year	42 mo
MAPK pathway status/mutation	BRAFV600E(PCR)	BRAFV600E(PCR)	pERK pos(IHC)	BRAFV600E(PCR)	BRAFV600E(PCR)
Treatment administered	Dabrafenib 5·25 mg/kg/day	Dabrafenib 5·25 mg/kg/day	Trametinib 0·025 mg/kg/day	Dabrafenib 5·25 mg/kg/day	Dabrafenib 5·25mg/kg/day
Treatment duration	4 mo (terminated)	6 mo (ongoing)	8 mo (ongoing)	6 mo (terminated)	7 mo (ongoing)
Comments	MAPKi stopped after 4 mo due to unclear inflammatory reaction			MAPKi stopped after 6 mo due to good therapy response
MRI changes on MAPKi	Unchanged ND findings, but new findings consistent with left mastoiditish	Pituitary stalk and pineal cyst normalized	Pituitary stalk normalized. Enhanced signal in globi pallidi reduced. Increased cerebellar atrophy.	Unchanged	Pituitary stalk and pineal gland normalized. Regression of enhanced T2 signal in dentate nuclei.
Clinical changes on MAPKi	Stable/unchanged	Stable/unchanged	Stable/unchanged	Marked improvement of behavioral problems. Neuropsychological tests improved.	More physically active. Neuropsychological evaluation unchanged.
Changes in academic level on MAPKi	Continues in normal schooling	Continues in normal schooling	Remains in special needs education	Academic difficulties were reduced. Now following regular schooling	Individualized plan in kindergarten. Improvement of delayed language and motor skills
Disease Activity Score at last follow‐upc	0	0	NA	0	0
Follow‐up time after initiation of MAPKi	11 mo	11 mo	11 mo	10 mo	9 mo

AD, active disease; Ara‐C, arabinoside cytosine; CNS, central nervous system; CsA, ciclosporin A; Dexa, dexamethasone; DI, diabetes insipidus; FLAIR, fluid‐attenuated inversion‐recovery; GH, growth hormone; HD, high‐dose; LCH, Langerhans cell histiocytosis; LN, lymph nodes; MAPKi, mitogen‐activated protein kinase inhibitor; MRI, magnetic resonance imaging; MS, multisystem; MTX, methotrexate; NA, not analyzed; N/A, not available; NAD, no active disease, ND, neurodegeneration; NFL, neurofilament light protein in CSF; PCR, polymerase chain reaction; pERK, phosphorylated extracellular‐signal‐regulated kinase; Pred, prednisolone; RO, risk organ; VBL, vinblastine; VCR, vincristine; 2‐CdA, cladribine; 6‐MP, 6‐mercaptopurine.

^aDiagnosed in another country, data refer to available information.

^bBone marrow involvement (CD1a‐pos cells) without haematopoietic involvement.

^cDisease Activity Score (DAS) according to Donadieu et al., 2004 (ref 13).

^dPosterior pituitary bright spot.

^eMRI one year after DI diagnosis.

^fEarliest available MRI at nine years of age.

^gNo signs of clinical or neuroradiological neurodegeneration according to current definitions.

^hIn addition fever, anaemia and elevated inflammatory parameters, but unclear if these were side effects or due to an infection.

Patients 1, 2, 4, and 5 had LCH with BRAF ^V600E mutation and were treated with dabrafenib 5·25 mg/kg/day. In patient 3, treated with trametinib 0·025 mg/kg/day, no BRAF ^V600E mutation was identified but staining for phosphorylated extracellular‐signal‐regulated kinase (ERK) was positive indicating an activated MAPK signaling pathway (Table I). ¹⁴ We also report routine CSF NFL levels in 12 additional children with LCH without evidence of ND‐CNS‐LCH (patient 6‐17, Supporting Table SII). The study was approved by the Ethics Review Board of Sweden (2019‐03956). Written informed consent was obtained for all five MAPKi‐treated patients.

CSF NFL levels, typically monitored three‐monthly after initiation of MAPKi, decreased markedly in all five children with CNS LCH (Fig 1). Within six months, CSF NFL had normalized (<380 ng/l) in four children and within nine months in all five (Fig 1, Supporting Table SI). In contrast, CSF NFL levels in patients 1 and 3 did not normalize on any other LCH‐directed chemotherapy prior to MAPKi (Supporting Tables SI and SII).

Fig 1.

Neurofilament (NFL) levels in the cerebrospinal fluid (CSF) in the five children with central nervous system (CNS) affection of Langerhans cell histiocytosis (LCH) before and after initiation of treatment with mitogen‐activated protein kinase pathway inhibitors (MAPKi). (A) Sequential neurofilament (NFL) levels in the CSF in patients 1–5. Duration of MAPKi therapy is indicated by the shaded area. The reference level (380 ng/l) is depicted as a dotted line. CSF NFL levels decreased markedly after initiation of treatment with MAPKi in all five children with CNS LCH. Within six months after treatment initiation, CSF NFL had normalized (<380 ng/l) in four children and within nine months in all five (see also Supporting Table SI). CSF NFL levels increased again after therapy cessation in patient 1 and 4. (B) The last CSF NFL value before initiation of MAPKi treatment is compared to the lowest CSF NFL value within nine months after initiation of MAPKi (P = 0·041, paired t‐test). The reference level (380 ng/l) is depicted as a dotted line.

Notably, CSF NFL levels increased again to abnormal levels within four months in the two patients whose MAPKi was discontinued (Fig 1, Supporting Table SI); in patient 1 due to an unclear inflammatory reaction and in patient 4 due to good response (see Supporting Text for details).

Among 16 CSF NFL samples from 11 additional children with LCH without known CNS LCH at sampling (patients 6–16, Supporting Table SII), only one was slightly elevated (440 ng/l). Of the three CSF samples from children with confirmed CNS LCH but without evidence of ND‐CNS‐LCH (patient 17 and patient 2 at DI diagnosis), only patient 2 had slightly elevated NFL (420 ng/l), which had been normal before DI. In contrast, CSF NFL levels were elevated in all 11 samples from the children with ND‐CNS‐LCH (Supporting Table SII).

No remarkable differences were observed for tau, phospho‐tau and GFAP in association with MAPKi or other therapies (Supporting Tables SI, SII). We did not encounter any severe complications in the 47 lumbar punctures performed.

MAPKi therapy was associated with perceivable neuroradiological and clinical improvement in three and two children, respectively (Table I). In patients 2, 3 and 5, a thickened pituitary stalk normalized and enlargement of the pineal cyst regressed. In patients 3 and 5, the enhanced signal in globi pallidi and dentate nuclei, respectively, was reduced (Supporting Figure S1). Patient 4 experienced impressive clinical improvement, behavioural problems disappeared, and neuropsychological tests improved, and patient 5 became more physically active. Limited adverse events were seen (fever, fatigue, myalgia in patient 1 and skin rash in patient 4).

Since clinical deterioration and the development of neuroradiological abnormalities are slow processes in CNS LCH, a surrogate marker to monitor CNS LCH neurodegeneration and therapy response is most valuable. We have previously reported an association between elevated CSF NFL levels and neurodegeneration in CNS LCH. ¹¹ , ¹² Based on the substantial amount of data on NFL in other neurodegenerative conditions, ¹⁰ as well as our data on CSF NFL levels in patients with and without ND‐CNS‐LCH (Supporting Tables SI, SII), it seems likely that CSF NFL actually reflects the extent of ongoing neurodegeneration also in LCH.

Principal limitations to our study are the small patient number and short follow‐up time. Nevertheless, with MAPKi treatment we noticed a remarkable normalization of CSF NFL levels not previously observed with other LCH‐directed therapies (Supporting Table SII). ¹² Similarly, CSF NFL levels were reduced in other diseases with specific disease‐modifying treatments (natalizumab in relapsing/remitting multiple sclerosis; nusinersen in spinal muscular atrophy). ¹⁰ , ¹⁵ One obvious drawback with MAPKi in CNS LCH is that the treatment likely has to be continued as long as clones with the disease‐causing oncogenic mutations remain, as illustrated by increasing CSF NFL levels in the two patients that discontinued dabrafenib therapy (Fig 1A, Supporting Table SI). ⁸ , ⁹

To conclude, we suggest prospective clinical trials in patients with or at risk of developing ND‐CNS‐LCH, with CSF NFL monitoring and, when appropriate, treatment with MAPKi and/or other relevant therapies, initiated early, preferably even before development of clinical or radiological signs of neurodegeneration. Relevant patients could be those with “CNS risk lesions”, multisystem disease, and known CNS involvement including endocrine deficiencies. ¹ The aim would be to reduce, prevent and ideally eliminate clinical neurodegeneration in LCH.

Author contributions

JIH conceived the study, consulted on patients, interpreted data, and drafted the manuscript. EK interpreted data, made figures, and assisted in drafting the manuscript. DMM reviewed MRIs and created figures. MCMK, BZ, TAN, CB, and IB treated patients and provided data. ML performed experiments and interpreted data. HZ and KB were responsible for analyses of neurodegenerative markers in patients 3–5. NH and DG interpreted data, assisted in drafting the manuscript, and DG also drafted Table SI. TvBG helped conceive the study, treated patients, consulted on patients, provided data, interpreted data, created Table I, and assisted in drafting the manuscript. JIH, EK, DG and TvBG verified the underlying data. All authors revised the manuscript critically for important intellectual content, had access to all the data in the study, and accept responsibility to submit for publication.

Conflict of interests

HZ has served at scientific advisory boards for Denali, Roche Diagnostics, Wave, Samumed, Siemens Healthineers, Pinteon Therapeutics and CogRx, has given lectures in symposia sponsored by Fujirebio, Alzecure and Biogen, and is a co‐founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program (outside submitted work). KB has served as a consultant, at advisory boards and at data monitoring committees for Abcam, Axon, Biogen, JOMDD/Shimadzu. Julius Clinical, Lilly, MagQu, Novartis, Roche Diagnostics, and Siemens Healthineers, and is a co‐founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program, all outside the submitted work. JIH has served as a consultant for Sobi, outside the submitted work. The other authors have no conflicts of interest to declare.

Supporting information

Table SI. Neurodegenerative biomarkers in the CSF in five children with CNS‐LCH in relation to MAPKi treatment.

Table SII. Neurodegenerative biomarkers in the CSF of 15 children with LCH without or prior to MAPKi treatment.

Table SIII. Description of analysis methods for the CSF biomarkers.

Fig S1. Neuroradiological findings prior to and after MAPKi therapy.