Supplementary Figure 1. Somatic mutations detected in histiocytic neoplasms. Number of

validated somatic mutations detected in pediatric and adult histiocytic neoplasm biopsies based

on whole exome sequencing.

2

Supplementary Figure 2. Frequencies of known activating kinase mutations in histiocytic

neoplasms. (a) Pie charts illustrating the frequencies of activating kinase alterations identified

by unbiased sequencing in Langerhans and non-Langerhans Cell Histiocytic neoplasms. (b) Pie

charts documenting the overall frequencies of kinase mutations from the BRAFV600E-wildtype,

non-LCH, FFPE tissue, validation cohort (left) and from the Erdheim-Chester Disease (ECD),

Juvenile Xanthogranuloma (JXG), and Rosai Dorfman Disease (RDD) subcategories of this

cohort (right 3 pie charts).

3

Supplementary Figure 3. ARAF mutations and variants of unknown significance detected

in BRAFV600E-wildtype, non-Langerhans Cell Histiocytic neoplasms. (a) IGV Browser

screen capture revealing ARAFS214A mutations in histiocyte femur biopsy lesion DNA but not

in peripheral blood DNA. (b) Hematoxylin and eosin stain (top) and immunohistochemical stain

for phosphorylated ERK in the femur biopsy of ARAFS214A- mutant ECD patient from (a) (400x

magnification; scale bar = 50 µm). (c) Gene diagram of definite somatic variants (blue font) and

variants of unknown significance (grey font) identified in WES, RNA-seq, and targeted

sequencing of entire coding region of ARAF across 44 histiocytic lesions. (d) List of variants

from diagram in (c) with details of clinical diagnosis and variant detected. Variants in blue text

are confirmed somatic.

4

Supplementary Figure 4. Clinical and histological images of the non-Langerhans cell

histiocytosis (non-LCH) lesions from index patients with kinase fusions identified by

RNA-seq. (a) Coronal brain MRI (left) of a 14-year-old patient with non-LCH brain histiocytic

infiltrates with the RNF11-BRAF fusion and the xanthogranulomatous histiocyte infiltration with

multinucleated giant cells (white arrow) in brain lesion (right) (H&E stain; 400x magnification;

scale bar = 50 µm). (b) Skin lesions (top) in a 25-year-old Erdheim-Chester Disease (ECD)

patient with KIF5B-ALK fusion with xanthogranulomatous histiocyte infiltration with Touton giant

cells (white arrows) (bottom) (H&E stain; 400x magnification; scale bar = 50 µm). (c)

Immunohistochemical staining (IHC) of ALK, pERK1/2, and CD68 in skin lesions with the

KIF5B-ALK fusion and a BRAFV600E-mutant, ECD patient (400x magnification; scale bar = 50

µm). (d) Skin lesions (top) in a 27-year-old ECD patient with an LMNA-NTRK1 fusion showing

5

xanthogranulomatous histiocyte infiltration (bottom) (H&E stain; 400x magnification; scale bar =

50 µm).

6

Supplementary Figure 5. Gene expression analysis of histiocytic neoplasms by RNA-seq.

(a) Gene sets differentially enriched in LCH (top) or non-LCH as detected by Gene Set

Enrichment Analysis (GSEA). (b) GSEA plots from the ranked list of differentially expressed

genes in LCH and non-LCH using 6 gene sets as displayed.

7

Supplementary Table 1. Characteristics of patient samples with histiocytic neoplasms used in this study and genomic analysis utilized for each sample.

Sample Diagnosis1 Patient Age Patient Sex Tissue Site Sequencing Method2 SJLCH02 LCH 1 Male Left Maxillary

Sinus WES and RNA-Seq

SJLCH04 LCH 2 Male Femur WES and RNA-Seq SJLCH05 LCH 8 Female Tibia WES and RNA-Seq SJLCH06 LCH 2 Male Skull WES SJLCH07 LCH 1 Male Epidural Space WES and RNA-Seq SJLCH08 LCH 4 Female Forehead Skin RNA-Seq SJLCH09 LCH 9 Male Scapula WES and RNA-Seq SJLCH13 JXG 14 Male Brain WES and RNA-Seq SJLCH14 LCH 10 Male Brain WES and RNA-Seq

MSKLCH14 ECD 78 Female Skin WES and RNA-Seq MSKLCH15-1 ECD 47 Female Bone WES and RNA-Seq MSKLCH15-2 ECD 47 Female Bone WES MSKLCH16 LCH 37 Female Bone WES MSKLCH17 ECD 75 Male Bone WES MSKLCH18 ECD 53 Male Kidney WES MSKLCH19 ECD 76 Female Skin WES MSKLCH20 ECD 65 Female Kidney WES MSKLCH21 LCH 72 Female Skin WES MSKLCH22 ECD 62 Male Skin WES MSKLCH23 ECD 68 Male Monocytes WES MSKLCH24 ECD 60 Male Kidney RNA-Seq MSKLCH25 ECD 61 Female Femur WES and RNA-Seq NIHECD6 ECD 50 Female Cerebellum WES NIHECD20 ECD 25 Female Skin WES and RNA-Seq

LMNA-NTRK1 ECD 27 Male Skin Targeted RNA-seq CLIP2-BRAF Non-LCH NOS 38 Female Retroperitoneum Targeted RNA-seq KIF5B-ALK ECD 50 Male Liver Targeted RNA-seq

Y11686 ECD 73 Male Peritoneum Targeted Sequencing3 Y121045 ECD 64 Male Pleura Targeted Sequencing Y12909 ECD 65 Male Pleura Targeted Sequencing Y11954 RDD 19 Female Lymph Node Targeted Sequencing Y11718 JXG 35 Male Skin Targeted Sequencing Y11719 JXG 22 Female Skin Targeted Sequencing Y12858 RDD 25 Female Lymph Node Targeted Sequencing Y13687 RDD 27 Male Lymph Node Targeted Sequencing Y11717 JXG 17 Female Skin Targeted Sequencing Y12531 JXG 23 Male Skin Targeted Sequencing Y11691 ECD 37 Male Bone NOS Targeted Sequencing Y11889 ECD 33 Male Bone NOS Targeted Sequencing Y11885 ECD 31 Male Tibia Targeted Sequencing Y11483 ECD 82 Female Skin Targeted Sequencing Y11850 ECD 55 Male Skin Targeted Sequencing

MD MEKQ56P ECD 55 Male Heart valve Targeted Sequencing Y12660 ECD 54 Male Mesentery Targeted Sequencing Y12663 ECD 52 Male Mesentery Targeted Sequencing Y11887 ECD 47 Male Skin Targeted Sequencing Y13161 ECD 71 Male Skin Targeted Sequencing Y11721 JXG 29 Male Skin Targeted Sequencing

8

Y11722 JXG 2 Male Skin Targeted Sequencing Y11725 JXG 63 Female Skin Targeted Sequencing

MSK IMPACT ECD 33 Male Tibial Bone Targeted Sequencing Y11849 RDD 49 Female Pituitary Stalk Targeted Sequencing Y13172 ECD 31 Male Paravertebral Targeted Sequencing Y11960 RDD 6 Male Cheek Targeted Sequencing Y11965 RDD 30 Female Meninges Targeted Sequencing Y13889 ECD 58 Male Perirenal Targeted Sequencing Y11720 JXG 27 Female Skin Targeted Sequencing Y11727 JXG 8 Male Skin Targeted Sequencing Y11750 RDD 62 Male Axillary Soft

Tissue Targeted Sequencing

Y11485 ECD 71 Male Retroperitoneal Adipose Tissue

Targeted Sequencing

Y11723 JXG 3 Male Skin Targeted Sequencing Y11728 JXG 39 Female Skin Targeted Sequencing Y101738 RDD 4 Female Lymph Node Targeted Sequencing Y14830 ECD 51 Male Skin; Peri-Renal Targeted Sequencing

1. ECD: Erdheim-Chester Disease; JXG: juvenile xanthogranuloma; LCH: Langherhans

cell histiocytosis; NOS: Not otherwise specified; RDD: Rosai Dorfman Disease. 2. WES: Whole exome sequencing. 3. Targeted sequencing refers to sequencing analysis of genomic DNA for the regions of

known mutations in NRAS, KRAS, BRAF, PIK3CA, and MAP2K1, as well as coding regions of ARAF.

9

Supplementary Table 2. Whole exome sequencing metrics.

WES Samples

Total Reads Mapped Reads

Duplicate Reads

Map % Dup % On-Target Bases

Mean Coverage

% Bases Above

10

% Bases Above

20

% Bases Above

50

% Bases Above

100 SJLCH02 95,246,934 93,956,134 6,513,689 99% 7% 61% 69.1 92.8% 89.4% 65.9% 19.1% SJLCH04 85,664,908 84,387,714 5,928,164 99% 7% 60% 61.9 91.6% 86.6% 57.4% 15% SJLCH05 86,945,644 85,330,749 9,464,196 98% 11% 64% 63.12 92.2% 87.5% 58.8% 15.2% SJLCH06 132,132,720 129,996,871 13,315,726 98% 10% 59% 90.24 93.6% 91.2% 78% 37.3% SJLCH07 113,038,788 110,992,843 12,016,235 98% 11% 62% 79.42 93.2% 90.3% 72.6% 27.9% SJLCH09 94,643,856 93,000,000 8,556,369 98% 9% 60% 65.75 92.4% 88.5% 62.4% 16.9% SJLCH13 120,351,454 118,035,196 12,187,853 98% 10% 61% 82.99 93.5% 90.9% 75.1% 30.8% SJLCH14 110,074,970 108,830,174 10,525,152 99% 10% 62% 78.31 93.8% 90.5% 68.7% 26.8%

MSKLCH14 120,741,520 117,813,736 6,866,425 98% 6% 62% 88.29 93% 90.2% 74.6% 35.3% MSKLCH15-1 113,627,942 111,044,348 7,099,432 98% 6% 63% 84.65 92.7% 89.6% 71.6% 32.3% MSKLCH15-2 160,222,570 155,742,470 10,445,675 97% 7% 64% 120.73 93.6% 91.6% 83.6% 57% MSKLCH16 142,266,008 137,840,856 9,388,364 97% 7% 64% 106.13 93.1% 90.8% 80.1% 48.4% MSKLCH17 170,907,176 166,240,452 11,261,935 97% 7% 64% 127.52 93.6% 91.7% 84.1% 59.5% MSKLCH18 113,289,452 111,599,145 9,749,512 99% 9% 59% 77.85 93.5% 91.0% 73.7% 26% MSKLCH19 91,683,260 90,593,665 6,931,399 99% 8% 60% 65.3 92.9% 89.6% 63.6% 15.4% MSKLCH20 107,180,094 105,877,069 9,201,691 99% 9% 60% 74.66 92.9% 89.9% 70.1% 24% MSKLCH21 85,338,734 84,186,594 6,426,411 99% 8% 61% 61.39 92.8% 89.3% 60.3% 12.1% MSKLCH22 97,016,410 95,733,732 7,930,774 99% 8% 58% 65.56 92.7% 88.6% 61% 16.9% MSKLCH23 135,135,058 133,079,037 12,954,751 98% 10% 57% 88.38 93.7% 91.5% 77.7% 35.5% MSKLCH25 148,214,701 147,793,213 13,040,911 99.7% 8.8% 61% 107.61 93.9% 91.8% 81.7% 49.1% NIHECD6 140,175,083 139,817,217 9,503,075 99.7% 6.8% 60% 101.56 94% 91.9% 80.6% 44.9%

NIHECD20 122,052,356 121,475,523 9,101,727 99.5% 7.5% 58% 86.58 93.1% 89.4% 67.8% 32.3%

10

Supplementary Table 3. Somatic variants identified by whole exome and whole transcriptome sequencing and validated by droplet-digital PCR and/or custom-capture targeted next-generation sequencing with variant allele frequencies (VAFs).

Case Gene Genomic Coordinates

Genomic Variant Amino Acid Variant VAFs

MSKLCH14 BRAF chr7:140453136 c.1799A>T p.V600E 5% MSKLCH14 GTF2F1 chr19:6381791 c.751_753del p.K251del 11%

MSKLCH15T1 BRAF chr7:140453136 c.1799A>T p.V600E 7% MSKLCH15T1 CSNK1G1 chr15:64508874 c.331G>T p.G111W 10% MSKLCH15T1 DDX43 chr6:74104738 c.110G>T p.R37L 8% MSKLCH15T1 DNMT3A chr2:25467132 c.1743G>T p.W581C 12% MSKLCH15T1 FAT1 chr4:187557879 c.3832G>T p.D1278Y 4% MSKLCH15T1 FGF5 chr4:81196141 c.434C>A p.S145X 6% MSKLCH15T1 JAK2 chr9:5069154 c.1459C>A p.R487S 5% MSKLCH15T1 MAP3K19 chr2:135756487 c.395G>T p.R132M 6% MSKLCH15T1 NCOA1 chr2:24933824 c.2443G>T p.D815Y 8% MSKLCH15T1 TAB3 chrX:30872481 c.1301C>A p.P434Q 4% MSKLCH15T1 TAB3 chrX:30873066 c.716C>A p.S239X 5% MSKLCH15T1 TRIO chr5:14474171 c.6048C>A p.F2016L 7% MSKLCH15T1 TTl1 chr20:36640424 c.1795G>T p.E599X 7% MSKLCH15T1 WDR33 chr2:128471212 c.3253G>T p.E1085X 8% MSKLCH15T1 ZFHX3 chr16:72992463 c.1582C>A p.Q528K 5% MSKLCH15T1 ZMYND8 chr20:45915981 c.721G>T p.E241X 5% MSKLCH15T2 BRAF chr7:140453136 c.1799A>T p.V600E 5% MSKLCH15T2 ARAF chrX:47430341 c.1616C>A p.P539H 2% MSKLCH15T2 AURKB chr17:8110666 c.226G>T p.D76Y 7% MSKLCH15T2 BCL2 chr18:60795947 c.631G>T p.D211Y 10% MSKLCH15T2 GAK chr4:887695 c.844G>T p.D282Y 6% MSKLCH15T2 MAPK9 chr5:179663476 c.1183G>T p.D395Y 7% MSKLCH15T2 STAT2 chr12:56737909 c.2101G>T p.E701X 6% MSKLCH15T2 YES1 chr18:743416 c.725-1G>T NA 5%

MSKLCH16 BRAF chr7:140453136 c.1799A>T p.V600E 13% MSKLCH16 ALK chr2:29917863 c.805G>T p.D269Y 9% MSKLCH16 AMER2 chr13:25745341 c.416_417insC p.G140fs 12% MSKLCH16 AURKB chr17:8110078 c.527G>T p.R176L 7% MSKLCH16 CCNT1 chr12:49108266 c.203G>T p.R68L 6% MSKLCH16 CHD7 chr8:61766944 c.6798G>T p.K2266N 7% MSKLCH16 CSNK1G1 chr15:64496755 c.884G>T p.R295L 5% MSKLCH16 CSPG4 chr15:75982702 c.704G>T p.R235L 6% MSKLCH16 DDX11 chr12:31254916 c.2202G>T p.K734N 7% MSKLCH16 DDX43 chr6:74115452 c.701G>T p.R234L 5% MSKLCH16 JAK2 chr9:5069158 c.1463C>A p.S488X 5% MSKLCH16 MAP3K9 chr14:71227812 c.908G>T p.R303L 5% MSKLCH16 MAPK11 chr22:50704972 c.679G>T p.D227Y 9% MSKLCH16 MUC4 chr3:195508598 c.9853G>T p.D3285Y 8% MSKLCH16 NCOA1 chr2:24952429 c.2946G>T p.L982F 5% MSKLCH16 RALGAPA1 chr14:36217952 c.1090G>T p.E364X 4% MSKLCH16 STAT6 chr12:57502001 c.61G>T p.D21Y 5% MSKLCH16 TTl1 chr20:36627633 c.2750G>T p.R917L 7% MSKLCH16 YES1 chr18:736929 c.1170G>T p.M390I 6% MSKLCH16 ZMYND8 chr20:45915981 c.721G>T p.E241X 5% MSKLCH17 BRAF chr7:140453136 c.1799A>T p.V600E 15% MSKLCH18 MAP2K1 chr15:66727455 c.171G>C p.K57N 7% MSKLCH18 CHD4 chr12:6692417 c.4007G>A p.G1336E 9% MSKLCH19 BRAF chr7:140453136 c.1799A>T p.V600E 9% MSKLCH19 ATXN1 chr6:16327948 c.593_594insGCA p.Q198delinsQQ 24% MSKLCH19 EP400 chr12:132547093 c.8181_8182insCAG p.Q2727delinsQQ 15%

11

MSKLCH19 HMGXB4 chr22:35661543 c.1163insA p.E388fs 10% MSKLCH19 KMT2C chr7:151962265 c.G1042G>A p.D348N 4% MSKLCH20 BRAF chr7:140453136 c.1799A>T p.V600E 12% MSKLCH20 FIP1L1 chr4:54319248 c.1447_1448del p.R483fs 11% MSKLCH20 TOX3 chr16:52502453 c.109G>A p.E37K 15% MSKLCH21 BRAF chr7:140453136 c.1799A>T p.V600E 7% MSKLCH21 ATRX chrX:76907782 c.4377_4379del p.E1459del 11% MSKLCH21 CHD4 chr12:6711207 c.355_357del p.K119del 17% MSKLCH21 CHD4 chr12:6682390 c.5407G>A p.A1803T 15% MSKLCH21 DNMT3B chr20:31395621 c.2474C>T p.A825V 12% MSKLCH21 FAT1 chr4:187541913 c.5827C>T p.Q1943X 6% MSKLCH21 GAK chr4:887704 c.835C>T p.P279S 8% MSKLCH21 MED12 chrX:70361098 c.6286_6288del p.Q2096del 11% MSKLCH21 MED15 chr22:20918817 c.532_534del p.Q178del 11% MSKLCH21 NCOA6 chr20:33334734 c.2793-2->T NA 27% MSKLCH21 SYNGAP1 chr6:33409086 c.2050G>A p.D684N 12% MSKLCH21 USP8 chr15:50784950 c.2287C>T p.R763W 16% MSKLCH21 WDR33 chr2:128467115 c.3514C>T p.R1172C 5% MSKLCH22 ARAF chrX:47426154 c.C674C>T p.A225V 3% MSKLCH22 AR chrX:66766390 c.1402delG p.G468fs 25% MSKLCH22 MUC4 chr 3:195508402 c.10049C>A p.T3350N 4% MSKLCH22 TAOK2 chr16:29994531 c.1138_1140del p.E380del 10% MSKLCH23 NRAS chr1:115256529 c.182A>G p.Q61R 50% MSKLCH23 ATXN1 chr6:16327915 c.626_627insGCA p.H209delinsQH 20% MSKLCH23 CHD7 chr8:61766036 c.6752C>T p.S2251L 47% MSKLCH23 GTF2F1 chr19:6381791 c.751_753del p.K251del 11% MSKLCH24 MAP2K1 chr15:66727486 c.202T>C p.F68L 11% MSKLCH25 ARAF chrX:47426120 c.640T>G p.S214A 8% MSKLCH25 CSPG4 chr15:75982058 c.1348T>A p.R450W 7% MSKLCH25 HUNK chr21:33312540 c.610+8T>- NA 13% MSKLCH25 INO80E chr16:30016629 c.602dupC p.T201fs 24% MSKLCH25 TRIO chr5:14388797 c.3948+9->T NA 11% NIHECD20 AFF3 chr2:100218011 c.1255_1257del p.S419del 11% NIHECD20 EP400 chr12:132539513 c.7729-6A>T NA 7% NIHECD20 INO80E chr16:30016629 c.602dupC p.T201fs 20% NIHECD20 HUNK chr21:33312540 c.610+8T>- NA 36% NIHECD20 RALGAPA1 chr14:36142064 c.3696+9A>C NA 17% NIHECD20 KMT2C chr7:151932897 c.2769+5G>T NA 6% NIHECD20 KMT2C chr7:151962113 c.1184+10C>A NA 4% NIHECD20 MAP4K4 chr2:102456430 c.923G>A p.R308H 30% NIHECD6 BRAF chr7:140453136 c.1799A>T p.V600E 19% NIHECD6 CSPG4 chr15:75982085 c.1321G>A p.E441K 10% NIHECD6 INO80E chr16:30016629 c.602dupC p.T201fs 12% NIHECD6 MED15 chr22:20918916 c.631_633del p.Q211del 10% NIHECD6 SETD8 chr12:123889486 c.713G>C p.R238P 9% NIHECD6 SETD8 chr12:123889492 c.A719C p.D240A 8% NIHECD6 USP8 chr15:50784990 c.2327T>C p.L776P 5% SJLCH02 BRAF chr7:140453136 c.1799A>T p.V600E 28% SJLCH02 CSPG4 chr15:75982085 c.G1321G>A p.E441K 7% SJLCH02 DIAPH chr5:140907266 c.3149-2->TTT NA 27% SJLCH02 KMT2C chr7:151932949 c.2722G>T p.G908C 5% SJLCH02 KRT1 chr12:53069223 c.1669_1689del p.S557_G563del 43% SJLCH02 PDS5B chr13:33344888 c.4161delA p.P1387fs 11% SJLCH02 TOX3 chr16:52473513 c.1338_1340del p.Q447del 15% SJLCH04 MAP2K1 chr15:66727443 c.159_173delTCTTACCCAGAAGCA p.F53_Q58del 15% SJLCH04 DIAPH chr5:140953564 c.1851_1853del p.P618del 20% SJLCH04 KRT1 chr12:53069189 c.1722_1723insGGC p.H575delinsGH 11% SJLCH04 MED15 chr22:20918916 c.631_633del p.Q211del 17% SJLCH04 NCOA6 chr20:33334734 c.2793-2->T NA 29%

12

SJLCH04 SYNGAP1 chr6:33419606 c.3956dupC p.A1319fs 14% SJLCH05 MAP2K1 chr15:66727457 c.169_183delCAGAAGGTGGGAGAA p.Q58_E62del 21% SJLCH05 AFF3 chr2:100218011 c.1255_1257del p.S419del 18% SJLCH05 FIP1L1 chr4:54319248 c.1447_1448del p.R483fs 15% SJLCH05 KRT1 chr12:53069223 c.1669_1689del p.S557_G563del 49% SJLCH06 MAP2K1 chr15:66727457 c.169_183delCAGAAGGTGGGAGAA p.Q58_E62del 10% SJLCH06 DKC1 chrX:154005089 c.1492_1494del p.K498del 13% SJLCH06 PLEKHG5 chr1:6529183 c.2397_2399del p.799_800del 16% SJLCH06 USP8 chr15:50784990 c.2327T>C p.L776P 5% SJLCH07 BRAF chr7:140453136 c.1799A>T p.V600E 11% SJLCH07 BRAF chr7:140453127 c.G1808C>T p.R603Q 13% SJLCH07 MED12 chrX:70361098 c.6286_6288del p.Q2096del 11% SJLCH07 MN1 chr22:28195603 c.927_929del p.Q309del 11% SJLCH07 PHF2 chr9:96439004 c.2961_2962insCCTGCCTCCACCACA p.T987delinsTPASTT 30% SJLCH08 MAP2K1 chr15:66727457 c.169_183delCAGAAGGTGGGAGAA p.Q58_E62del 23% SJLCH09 BRAF chr7:140453136 c.1799A>T p.V600E 14% SJLCH09 ATXN1 chr6:16327915 c.626_627insGCA p.H209delinsQH 20% SJLCH09 EP400 chr12:132547068 c.8156_8157insGCA p.R2719delinsRQ 39% SJLCH09 GTF2F1 chr19:6381410 c.976_978del p.E326del 15% SJLCH09 HMGXB4 chr22:35661543 c.1163dupA p.E388fs 11% SJLCH09 MED15 chr22:20918916 c.631_633del p.Q211del 16% SJLCH09 MN1 chr22:28194913 c.1617_1619del p.Q539del 10% SJLCH09 PHF2 chr9:96439004 c.2961_2962insCCTGCCTCCACCACA p.T987delinsTPASTT 48% SJLCH09 THAP11 chr16:67876824 c.367_369del p.Q123del 19% SJLCH13 DDX11 chr12:31244665 c.1102C>T p.P368S 8% SJLCH13 KMT2C chr7:151945330 c.2189C>A p.S730Y 11% SJLCH13 MED12 chrX:70361098 c.6286_6288del p.Q2096del 12% SJLCH13 MUC4 chr3:195507020 c.11431G>C p.V3811L 10% SJLCH14 BRAF chr7:140453136 c.1799A>T p.V600E 9% SJLCH14 AR chrX:66765159 c.171_173del p.Q58del 14% SJLCH14 AR chrX:66766356 c.1368_1369insGGC p.G456delinsGG 25% SJLCH14 CCNT1 chr12:49087434 c.1561_1563del p.H521del 15%

13

Supplementary Table 4: List of the top 1% of differentially expressed genes across histiocytic samples from mRNA sequencing. Top 1% of Differentially Expressed Genes (159)

MT1G MT1H HBA2 HBA1 EMR1 IGLL5

HLA-DRB6 CCL4L1

ATP6VOD2 ACP5

APOC2 ITGB3 CTSK

AKAP6 HIF1A-AS2

IL8 EGFL6 MMP9

TNFAIP6 MMP13

SCARNA3 HERC2P10

FOS RGS1

SCARNA20 CYR61

FN1 SIGLEC1

SIGLEC11 MS4A1 BANK1 CXCL13 CCL19 RHOH IL7R

TLR10 CD89

WNT5A CD24

EIF1AY TXLNG2P RPS4Y1 DDX3Y PRKY

KDM5D KCNN4 GPR84

CLEC5A IL1B

14

Top 1% of Differentially Expressed Genes (159) CDKN2A SMYD3 ADAM 8

ANKRD36BP1 GHRL DUSP4

AGPAT9 CD1C S100B IL18R1 CCR6

SUSD3 PPM1N

TNF ITGB1BP2 LGALS12 IL22RA2

EMR3 C21orf67 NDRG2

TACSTD2 RASAL1

HLA-DQB2 HIST1H2AL HIST1H3J HIST1H2AI HIST1H3B HIST1H3G HIST1H4A HIST1H3F FAM109A

PLEK2 PKIB CD1E

MMP12 CD1A

HIST1H2BO HIST1H2AB HIST1H2BI HIST1H2BM

MMP1 PTGS2

C15orf48 CD207

SYNPO2 PALMD

IL33 MTRNR2L2 MTRNR2L8

AQP1 CXCL12 CCL14

15

Top 1% of Differentially Expressed Genes (159) MAF

STEAP4 NFIA

TGFBR3 ME1

MPDZ MGP H19

CCDC152 SEPP1

SLC40A1 CXCL9 ABI3BP ABCA6

CFD GGTA1P

RARRES1 DHCR24 TPSAB1 CLDN1 HOPX

PTGER3 TMEM45A

SDC1 CDR1 SLP1 NBL1

IGFBP5 KLF4

SCARNA8 SCARNA4

HTRA1 FBLN1 FBN1

OLFML3 SFRP4 AEBP1

FAP THBS2

LRRC15 SCARA5 HOMER3 CPXM1 VSIG4 TREM2

FCGR3A MSR1 CCL8

C3 F13A1 LYVE1 MRC1

16

Top 1% of Differentially Expressed Genes (159) CCDC80 SEMA3C FOLR2

RNASE1 PTN KLF2

17

Supplementary Table 5: PCR primers with M13F2 and M13R2 tails (blue) for Sanger sequencing used in the targeted sequencing recurrence testing for MAP2K1 exons 2 and 3 and all coding regions of ARAF.

Gene Exon Primer Type Primer Sequence MAP2K1 2 Forward GTAAAACGACGGCCAGTGACTTGTGCTCCCCACTTT MAP2K1 2 Reverse CAGGAAACAGCTATGACCGTCCCCAGGCTTCTAAGTACC MAP2K1 3 Forward GTAAAACGACGGCCAGTTCATCCCTTCCTCCCTCTTT MAP2K1 3 Reverse CAGGAAACAGCTATGACCCTCTTAAGGCCATTGCTCCA

ARAF 2 Forward GTAAAACGACGGCCAGTACAGGAGCCTGGACTTTGAA ARAF 2 Reverse CAGGAAACAGCTATGACCTCACCCTTTTGATCCCAGAG ARAF 3 Forward GTAAAACGACGGCCAGTCTTTGCAGAAGGATGGGAAC ARAF 3 Reverse CAGGAAACAGCTATGACCTCAAATGACTGAGGAGGGGA ARAF 4 Forward GTAAAACGACGGCCAGTGACATGACCCTGTGGGTAGC ARAF 4 Reverse CAGGAAACAGCTATGACCTACCTGATGCCACAGAGCAG ARAF 5 Forward GTAAAACGACGGCCAGTATTTTGTGAGTGCAGGGTGG ARAF 5 Reverse CAGGAAACAGCTATGACCCAGCTGTCTGTAAGGGCCTC ARAF 6 Forward GTAAAACGACGGCCAGTATGGGGAGCACAGAGGC ARAF 6 Reverse CAGGAAACAGCTATGACCGTGGTCTACAAGAGCCCCAA ARAF 7 Forward GTAAAACGACGGCCAGTAATGCATGTTTATGGCTGGG ARAF 7 Reverse CAGGAAACAGCTATGACCGTCAAAAAGAGGGCATGGAG ARAF 8 Forward GTAAAACGACGGCCAGTACCACAGGGCAGAGGGTAG ARAF 8 Reverse CAGGAAACAGCTATGACCTGCAGAGAGGATGTGAGTGG ARAF 9 Forward GTAAAACGACGGCCAGTTCAGCACTGATGGTGAGTCC ARAF 9 Reverse CAGGAAACAGCTATGACCCCAGATGGGTGGCATCTAAG ARAF 10 Forward GTAAAACGACGGCCAGTACCAAGGCTGAGTGACGTG ARAF 10 Reverse CAGGAAACAGCTATGACCTTTCCCAGAGAGATGCCAAG ARAF 11 Forward GTAAAACGACGGCCAGTTTTTCCTCCCCACCTCTGA ARAF 11 Reverse CAGGAAACAGCTATGACCCGGTGGATGATGTTCTTGG ARAF 12 Forward GTAAAACGACGGCCAGTGAGTGAGCCTCCCAGCCT ARAF 12 Reverse CAGGAAACAGCTATGACCATGCCCCTCCCTTGTAATTC ARAF 13 Forward GTAAAACGACGGCCAGTGGATGAGTGGTATAGGGGCA ARAF 13 Reverse CAGGAAACAGCTATGACCCACATCTGGCACAGGCATC ARAF 14 Forward GTAAAACGACGGCCAGTTGAGTTGTACCCTGTGTGGC ARAF 14 Reverse CAGGAAACAGCTATGACCTTATCAGTGCAAAGGAGGGG ARAF 15 Forward GTAAAACGACGGCCAGTCCCAGCTCGCTAAAAATGAA ARAF 15 Reverse CAGGAAACAGCTATGACCTGCCTCCATCTCAGTCTTCC ARAF 16 Forward GTAAAACGACGGCCAGTCCCATGTGCTTTTCCAGTTC ARAF 16 Reverse CAGGAAACAGCTATGACCCAAGAGAAGAGTCGGGAAACA

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Supplementary Methods

Interphase Fluorescence in situ Hybridization (FISH)

A commercially available break-apart probe was used for ALK according to the manufacturer’s

protocol (Abbott, Des Plaines, IL). For BRAF (1) and NTRK1 (2) fusions, break-apart probes

were prepared as described previously.

The probes were hybridized on 5µm-thick tissue sections, and the number and

localization of the hybridization signals were assessed in a minimum of 100 interphase nuclei

with well-delineated contours, and at least 50% neoplastic cells had to show a split signal to

report a rearrangement of a kinase.

Dual-color FISH was performed on 4µm-thick, FFPE tissue sections. BAC clones

(BACPAC Resources, Oakland, CA) were used to develop a break-apart FISH probe for the

BRAF gene (RP11-96I22 + RP11-837G3, labeled with AlexaFluor-488 & RP11-948O19, labeled

with AlexaFluor-555). Probes were co-denatured with the target cells on a slide moat at 90°C for

12 minutes. The slides were incubated overnight at 37°C on a slide moat and then washed in

4M Urea/2xSSC at 25°C for 1 minute. Nuclei were counterstained with DAPI (200ng/ml)(Vector

Labs). Images were captured and processed using the Cytovision v7.3 software from Leica

Biosystems (Richmond, IL).

Immunohistochemistry

Immunohistochemical analysis was performed on archival, FFPE tumor specimens to confirm

the diagnosis and subtype of histiocytic neoplasm, expression of the fusion kinases, and the

activation of oncogenic pathways. We used a Discovery Ultra instrument with a multimer/DAB

detection system (Ventana Medical Systems Inc., Tucson, AZ, USA) with appropriate negative

and positive controls using the following antibodies: ALK (clone D5F3; Cell Signaling, Danvers,

MA, USA; dilution: 1:250), NTRK1 (clone EP1058Y; Epitomics, Burlingame, CA, USA; dilution:

1:100), CD68 (clone: KP1; DAKO, Carpinteria, CA, USA; dilution: 1:2000), phospho-ERK1/2,

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(clone: Rabbit MAb; Cell Signaling Technologies, Danvers, MA, USA; 1:1000), and CD163

(clone: 10D6; Vector, Burlingame, CA, USA; dilution: 1:200). The percentage of tumor cells

exhibiting staining was scored by at least two independent pathologists. Microscopic slides were

evaluated using an OLYMPUS BX41 microscope (Olympus Scientific Solutions, Waltham, MA,

USA), and images were acquired using an OLYMPUS DP72 camera (Olympus Scientific

Solutions, Waltham, MA, USA).

Plasmids

To investigate the functional roles and the activation of oncogenic signaling pathways, we

cloned the RNF11-BRAF and KIF5B-ALK fusions and expressed them in Ba/F3 cells. MSCV-

based expression vectors with GFP and the full-length BRAF wild type and BRAFV600E were

used as controls. Fusion constructs were cloned into the MSCV-IRES-GFP backbone and

checked by digestion and sequencing. For MEK1 functional experiments, MEK1 mutations were

introduced into MEK1 PLASMID using the site-directed Mutagenesis Kit (Stratagene). HA-ERK2

in BACKBONE was also used.

Western Blotting

Anti-ALK (no. 3791), anti-phospho-ALK (Tyr1096) (no. 6962), anti-phospho-p44/42 MAPK

(ERK1/2) (Thr202/Tyr204) (no. 9101), anti-AKT (no. 9272), anti-phospho-AKT (Ser473) (no.

9271), anti-STAT3 (79D7) (no. 4904), anti-phospho-STAT3 (Tyr705) (no. 9131), as well as the

secondary antibodies anti-rabbit IgG-HRP (no. 7076) and anti-mouse IgG-HRP (no. 7074) were

purchased from Cell Signaling Technology. Anti-KIF5B [EPR10276(B)] (ab167429) was

purchased from abcam®. Anti-β-Actin (A5441) was purchased from Sigma-Aldrich®.

Cell lysates were prepared in RIPA buffer supplemented with Halt protease and

phosphatase inhibitor cocktail (Thermo Scientific). Equal amounts of protein, as measured by

the BRADFORD protein assay, were resolved in 4–12% Bis-Tris NuPage gradient gels (Life

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Technologies) and transferred electrophoretically on a polyvinylidene difluoride 0.45-m

membrane. Membranes were blocked for 1 h at room temperature in 5% bovine serum albumin

(BSA) in TBST before being incubated overnight at 4°C with the primary antibodies. All primary

antibodies were diluted 1:1,000 in 5% BSA in TBST, except anti-β-actin, which was diluted

1:5,000 in 5% BSA in TBST. After three washes of 5 min in TBST, secondary antibodies were

diluted 1:2,000 in 5% BSA in TBST and incubated for 1 h at room temperature. After another

three washes in TBST, detection of the signal was achieved by incubating the membrane on an

ECL solution from Millipore and exposure on autoradiography films from Denville Scientific

(Metuchen, NJ, USA).

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References

1. Zhang J, Wu G, Miller C, Tatevossian R, Dalton J, Tang B, et al. Whole-genome sequencing identifies genetic alterations in pediatric low-grade gliomas. Nature genetics. 2013;45:602-12. 2. Wiesner T, He J, Yelensky R, Esteve-Puig R, Botton T, Yeh I, et al. Kinase fusions are frequent in Spitz tumours and spitzoid melanomas. Nature communications. 2014;5:3116.