Mass spectrometric genotyping (OncoMap) Sensitivity of genotyping Laser capture microdissection Quantifying Langerhans cells in LCH samples

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1 Mass spectrometric genotyping (OncoMap) DNA was quantified using picogreen analysis, then subjected to whole genome amplification and used for further analysis only if DNA fingerprinting demonstrated non-biased amplification. Whole genome amplified DNA was used as input for multiplex PCR using primers from OncoMap 3 and OncoMap 3 Extended which together comprise 1047 independent assays interrogating 983 unique mutations across 115 genes (Tables S6 and S7). 1 Single base primer extension was performed using iplex Gold single base extension enzyme (Sequenom, San Diego, CA) and products were transferred to SpectroCHIPs for analysis by MALDI-TOF mass spectrometry. Allele peaks were flagged using a modified Sequenom algorithm followed by manual review by two independent reviewers of candidate calls which were classified as aggressive or conservative depending on their apparent robustness. Sample quality was considered adequate for analysis if more than 80% of the attempted genotypes resulted in identifiable products. Candidate mutations were validated using multi-base hme extension chemistry and a bidirectional assay design interrogating both DNA strands independently from unamplified genomic DNA. 1 Validation was performed for the following alleles: BRAF V600E, BRAF V600_K601>E, TP53 R175H, MET E168D, EGFR S703F, and CUBN I3189V. Proportion of mutant alleles in each sample was determined by dividing the area of the mutant allele peak identified in hme by the sum of the areas of the mutant and wild type allele peaks. Sensitivity of genotyping Our data indicate that the abundance of mutant alleles is directly proportional to the abundance of pathologic Langerhans cells in the cored sample (Fig. S1). The percentage of mutant alleles never exceeded 50% even in microdissected samples suggesting that the mutant allele may be present in one heterozygous copy per cell (although this is speculative). Finally, pyrosequencing requires an abundance of >4% mutant alleles to call a sample mutant. Based on these considerations, a clinical sample would have to contain at least 4% pathologic Langerhans cells carrying mutant BRAF in order to be called mutant in this study. Laser capture microdissection Seven micron thick sections were placed on PEN Membrane glass slides (Molecular Devices, Sunnyvale, CA). Sections were deparaffinized, stained with Histo-green, and dehydrated. Cell types were identified based on morphology and captured using a Veritas Microdissection system (Molecular Devices). DNA was extracted using the QIAmp DNA FFPE tissue kit as described above. Quantifying Langerhans cells in LCH samples For each sample, Langerhans cells in a microscopic field corresponding to the location of core sample were identified based on nuclear morphology and manually counted as were all cell types in order to derive a percentage of Langerhans cells in the field. This process was repeated for 3-5 microscopic fields and an average percentage of Langerhans cells was derived for each sample. Fluorescence in situ hybridization Four micron tissue sections were prepared on glass slides as described. 2 The following DNA probes were co-hybridized: RP11-767F15 (SpectrumGreen), which maps to 7q34, 5' of BRAF; RP11-60F17 (SpectrumOrange), which maps to 7q34, 3' of BRAF; and the D7Z1 control probe (SpectrumAqua), which maps to 7p11.11-q The D7Z1 probe was purchased pre-labeled

2 from Abbott Molecular/Vysis, Inc. (Des Plaines, IL). The BRAF-flanking BAC clones were obtained from CHORI ( DNA probes were direct-labeled using nick translation according to standard protocols. Hybridization and imaging were performed as described. 2 Immunohistochemistry Immunohistochemical staining using anti-phospho-mek and anti-phospho-erk antibodies (rabbit IgG from Cell Signaling Technology, Inc., Danvers MA) was optimized using a Ventana Discovery XT automated immunohistochemistry slide processing platform according to manufacturer s instructions (Ventana Medical Systems, Tucson, AZ). Standard quality control procedures were undertaken to optimize antigen retrieval, primary antibody dilution, secondary antibody detection and other factors for both signal and noise. CD1a staining was performed using monoclonal antibody clone MTB1 (Ventana Medical Systems) and standard procedures. A semi-quantitative staining score was derived for each sample by comparing its staining intensity to that of Langerhans cells in dermatopathic lymphadenopathy samples (normal Langerhans cells in skin were designated as 0) and to the most intensely stained LCH and dermatopathic lymphadenopathy samples (designated as 3+). This was repeated for 10 microscopic fields and an average staining intensity was inferred. Microscopy was performed using an Olympus BX-51 Research Microscope (Olympus America, Center Valley, PA) with a U-Plan SApo 100x objective having a numerical aperture of All imaging work was done at room temperature using oil immersion, and images were captured with a CMAD-3 camera (Olympus America) and Cytovision acquisition software (Genetix USA, Boston, MA). REFERENCES 1. MacConaill LE, Campbell CD, Kehoe SM, Bass AJ, Hatton C, Niu L, et al. Profiling critical cancer gene mutations in clinical tumor samples. PLoS ONE 2009; 4: e Firestein R, Bass AJ, Kim SY, Dunn IF, Silver SJ, Guney I, et al. CDK8 is a colorectal cancer oncogene that regulates beta-catenin activity. Nature 2008; 455:

3 Table S1. BRAF mutational status in dermatopathic lymphadenopathy and Rosai-Dorfman disease Patient Diagnosis % BRAF Mutant % BRAF Mutant OncoMap % Alleles Alleles BRAF Histiocytes Pyrosequencing Pyrosequencing LCM Status* Whole Samples Material DL1 Dermatopathic 30% nt 3.81% nt Lymphadenopathy DL2 Dermatopathic 45% nt 2.01% nt Lymphadenopathy DL3 Dermatopathic 40% nt 1.32% 2.38% Lymphadenopathy DL4 Dermatopathic 30% nt 2.40% nt Lymphadenopathy DL5 Dermatopathic 40% nt 2.99% 2.76% Lymphadenopathy RDD1 Rosai-Dorfman 60% WT nt nt Disease RDD2 Rosai-Dorfman 30% WT nt nt Disease RDD3 Rosai-Dorfman 65% WT nt nt Disease RDD4 Rosai-Dorfman Disease 40% WT 2.05% nt a BRAF alleles present in samples as determined by hme. nt, not tested; WT, wild type b Proportion of BRAF V600E mutant sequences determined by pyrosequence analysis of cores taken from whole tissue blocks. Samples with < 4% mutant alleles are considered to be uniformly wild type. nt, not tested c Proportion of BRAF V600E mutant sequences determined by pyrosequencing analysis of Langerhans cell-rich regions of tissue obtained by laser-capture microdissection (LCM). Samples with < 4% mutant alleles are considered to be uniformly wild type. nt, not tested

4 Table S2. Clinical Characteristics by Mutational Status Total N (%) BRAF V600E Mutation Present N (%) BRAF V600E Mutation Absent N (%) p-value* Number of Pts (57) 26 (43) Age (yrs), median (range) 12 (0.75, 61) 9 (0.75, 61) 31 (1, 61) 0.03 Age <10 years 25 (41) 19 (56) 6 (24) years 34 (56) 15 (44) 19 (76) Unknown 2 (3) Sex Males 38 (62) 24 (71) 14 (56) 0.28 Females 21 (34) 10 (29) 11 (44) Unknown 2 (3) Disease Site Bone 37 (61) 22 (65) 15 (58) 0.54 Lung 12 (20) 5 (15) 7 (27) Other 11 (18) 7 (21) 4 (15) Soft Tissue 4 (7) Lymph Node 3 (5) Thymus/Thyroid 2 (3) Skin 1 (2) Mediastinum 1 (2) Unknown 1 (2) Stage 47 (77) 28 (88) 19 (83) 0.71 Single System 8 (13) 4 (12) 4 (17) Disseminated 6 (10) *Fisher s exact test for categorical comparisons or the Wilcoxon rank-sum test for differences between continuous measures (age). Data indicated as unknown are excluded from testing.

5 Table S3. Unadjusted (Univariate) and Adjusted (Multivariable) Exact Logistic Regression Models to Examine BRAF V600E Mutational Status and Clinical Characteristics* Clinical Characteristic BRAF V600E BRAF V600E Unadjusted Mutation Mutation Adjusted Exact Exact p-value Present Absent OR (95% CI) OR (95% CI) N (%) N (%) p-value N Age (yrs.) b, median (range) 9 (0.75, 61) 31 (1, 61) 0.97 ( ) ( ) 0.09 Age <10 yrs. 19 (76) 6 (24) 3.91 ( ) ( ) yrs. 15 (44) 19 (56) Sex Female 10 (48) 11 (52) 0.54 ( ) ( ) 0.31 Male 24 (63) 14 (37) Disease Site Bone 22 (59) 15 (41) 0.84 ( ) > ( ) 0.62 Lung 5 (42) 7 (58) 0.43 ( ) ( ) >0.99 Other 7 (64) 4 (36) Stage Single System 28 (60) 19 (40) 1.46 ( ) ( ) 0.43 Disseminated 4 (50) 4 (50) *Calculations for the Unadjusted OR for age, sex and stage included either 59 or 55 patients as noted and only the 55 patients with all clinical characteristic data could be included in the adjusted model. Reference group does not have OR listed N=59 for unadjusted models with age and sex, N=60 for unadjusted model with disease site, N=55 for unadjusted model with stage N=55 and the results listed for sex, disease site and stage include age dichotomized in the adjusted mode

6 Table S4. BRAF mutational status in laser-capture microdissected material from LCH cases* Patient b % Mutant Allele (Unfractionated) % Mutant Allele (LC-Enriched) % Mutant Allele (LC-Depleted) Fold Enrichment in LC Fraction % 6.56% 2.27% % 22.38% 2.90% % 47.34% 2.22% % 31.59% 5.23% 6.0 * Proportion of BRAF V600E mutant sequences determined by pyrosequencing analysis of the indicated samples. Patient numbers correspond to those in Table 1. LC, Langerhans cell

7 Table S5. Immunohistochemical staining of LCH cases for p-mek and p-erk* Patient p-mek p-erk 2 1+/2+ 2+/ / /2+ 5 0/ / / / / / /1+/ /2+ 0/ /3+ 2+/ /3+ 2+/ /3+ 2+/ / / / / / / / / /2+ 1+/2+ 59 DL1 1+/2+ 2+ DL DL DL4 1+/2+ 2+ DL *Staining intensities are shown on a 0 to 3+ scale in which normal Langerhans cells in skin were scored as 0 and the most intensely stained LCH and dermatopathic lymphadenopathy cases were scored as 3+. Multiple values indicate staining intensities in separate areas of the sample. Blank entries indicate that there was insufficient material for analysis. Patient sample numbers correspond to those in Table 1. DL1-5 are samples from patients with dermatopathic lymphadenopathy.

8 Table S6. OncoMap 3 Core Assays* GENE_SYMBOL AA_CHANGE GENE_SYMBOL AA_CHANGE GENE_SYMBOL AA_CHANGE GENE_SYMBOL AA_CHANGE ABL1 D276G CTNNB1 S33Y HRAS Q61K NRAS G13R ABL1 E255K CTNNB1 S33Y HRAS Q61L NRAS G13S ABL1 E255V CTNNB1 S37A HRAS Q61L NRAS G13V ABL1 E355G CTNNB1 S37C HRAS Q61P NRAS G13V ABL1 F317L CTNNB1 S37F HRAS Q61R NRAS Q61E ABL1 F359V CTNNB1 S37P HRAS Q61R NRAS Q61H ABL1 G250E CTNNB1 S37Y JAK2 V617F NRAS Q61H ABL1 H396R CTNNB1 S45A JAK2 V617F NRAS Q61K ABL1 L248V CTNNB1 S45C JAK3 A572V NRAS Q61K ABL1 M244V CTNNB1 S45F JAK3 P132T NRAS Q61L ABL1 M351T CTNNB1 S45F JAK3 V722I NRAS Q61L ABL1 Q252H CTNNB1 S45P KIT D52N NRAS Q61P ABL1 Y253F CTNNB1 S45Y KIT D816H NRAS Q61R ABL1 Y253H CTNNB1 T41A KIT D816V NRAS Q61R AKT1 E17K CTNNB1 T41I KIT D816V PDGFRA D842_D846>E AKT2 R371H CTNNB1 T41P KIT D816Y PDGFRA D842_D846>G AKT2 S302G CTNNB1 T41S KIT D816Y PDGFRA D842_D846>N APC E1306* CTNNB1 V22_G38del KIT E839K PDGFRA D842_H845>V APC E1309fs*4 CTNNB1 V22A KIT E839K PDGFRA D842_H845del APC E1309fs*6 CTNNB1 W25_D32del KIT K550_K558del PDGFRA D842_M844del APC E1379* EGFR A750P KIT K558_E562del PDGFRA D842_S847>EA APC Q1338* EGFR D761N KIT K558_V560del PDGFRA D842I APC Q1367* EGFR D761Y KIT K642E PDGFRA D842V APC Q1378* EGFR D770_N771insG KIT L576P PDGFRA D842Y APC Q1429* EGFR D770_N771insN KIT N822K PDGFRA D842Y APC R1114* EGFR E734K KIT N822K PDGFRA D846Y APC R1450* EGFR E746_A750>V KIT P585P PDGFRA I843_D846del APC R1450* EGFR E746_A750del KIT T670I PDGFRA I843_S847>T APC R876* EGFR E746_S752>A KIT V559A PDGFRA R841_D842del APC S1465fs*3 EGFR E746_S752>D KIT V559D PDGFRA S566_E571>K APC T1667fs*9 EGFR E746_T751>A KIT V559del PDGFRA S566_E571>R BRAF D587A EGFR E746_T751del KIT V559G PDGFRA S566_E571>R BRAF D587E EGFR E746K KIT V559I PIK3CA C420R BRAF D594E EGFR G719A KIT V560D PIK3CA E542K BRAF D594G EGFR G719C KIT V560G PIK3CA E542K BRAF D594V EGFR G719D KIT V654A PIK3CA E545A BRAF E586K EGFR G719S KIT V825A PIK3CA E545G BRAF F595L EGFR G735S KIT W557G PIK3CA E545K BRAF F595S EGFR G810D KIT W557R PIK3CA E545K BRAF G464E EGFR G810S KIT W557R PIK3CA H1047L BRAF G464R EGFR H773R KIT Y503_F504insAY PIK3CA H1047R BRAF G464V EGFR K745R KRAS A146T PIK3CA H1047R BRAF G466A EGFR L730F KRAS A146T PIK3CA H1047Y BRAF G466E EGFR L747_A750>P KRAS A59T PIK3CA H701P BRAF G466R EGFR L747_E749del KRAS G12A PIK3CA N1068fs*4 BRAF G466V EGFR L747_P753>Q KRAS G12A PIK3CA N345K BRAF G466V EGFR L747_P753>S KRAS G12C PIK3CA P539R BRAF G469A EGFR L747_R748>FP KRAS G12C PIK3CA R88Q BRAF G469E EGFR L747_S752del KRAS G12D PIK3CA Y1021C BRAF G469R EGFR L747_T751>P KRAS G12D PTEN K267fs*9 BRAF G469R EGFR L747_T751>S KRAS G12R PTEN K6fs*4 BRAF G469S EGFR L747_T751del KRAS G12R PTEN N323fs*2 BRAF G469V EGFR L858M KRAS G12S PTEN N323fs*21 BRAF G596R EGFR L858M KRAS G12S PTEN P248fs*5 BRAF I592M EGFR L858R KRAS G12V PTEN R130* BRAF I592V EGFR L858R KRAS G12V PTEN R130fs*4 BRAF K601del EGFR N771_P772>SVDNR KRAS G13A PTEN R130G BRAF K601E EGFR p.e746_a750del KRAS G13A PTEN R130Q BRAF K601N EGFR P733L KRAS G13C PTEN R173C BRAF L597Q EGFR P753S KRAS G13C PTEN R173H BRAF L597R EGFR P772_H773insV KRAS G13D PTEN R233* BRAF L597S EGFR S752_I759del KRAS G13D PTEN R335* BRAF L597V EGFR S752_I759del KRAS G13R PTEN V317fs*3 BRAF N581S EGFR S752Y KRAS G13R RB1 C706F BRAF R444W EGFR T790M KRAS G13S RB1 E137* BRAF S605F EGFR T790M KRAS G13S RB1 E748* BRAF S605N EGFR V742A KRAS G13V RB1 L199* BRAF T599_V600insTT EGFR W731* KRAS G13V RB1 L660fs*2 BRAF T599I ERBB2 D769H KRAS L19F RB1 L660fs*2 BRAF V471F ERBB2 G776S KRAS L19F RB1 R320* BRAF V600A ERBB2 G776VC KRAS Q22K RB1 R358* BRAF V600D ERBB2 L755P KRAS Q22K RB1 R455* BRAF V600E ERBB2 L755S KRAS Q61E RB1 R552* BRAF V600L ERBB2 V777L KRAS Q61E RB1 R556* BRAF V600M ERBB2 V777L KRAS Q61H RB1 R579* BRAF V600R FGFR1 P252T KRAS Q61H RET A883F CDK4 R24H FGFR1 S125L KRAS Q61H RET C634R CDKN2A D108Y FGFR2 C382R KRAS Q61H RET C634W CDKN2A D84Y FGFR2 K310R KRAS Q61K RET C634Y CDKN2A E61* FGFR2 N549K KRAS Q61K RET D631_L633>E CDKN2A E61* FGFR2 N549K KRAS Q61L RET D631G CDKN2A E69* FGFR2 S252W KRAS Q61L RET D898_E901del CDKN2A E69* FGFR2 S372C KRAS Q61P RET E632_A640>VRP CDKN2A E88* FGFR2 Y375C KRAS Q61P RET E632_L633>V CDKN2A E88* FGFR3 G370C KRAS Q61R RET E632_L633del CDKN2A H83Y FGFR3 G370C KRAS Q61R RET E768D CDKN2A H83Y FGFR3 G697C MET H1112R RET F612_C620del CDKN2A P114L FGFR3 K650Q MET H1112Y RET M918T CDKN2A R58* FGFR3 L794fs*23 MET M1268T SRC Q531* CDKN2A R80* FGFR3 R248C MET T1010I STK11 D194N CDKN2A R80* FGFR3 R248C MET Y1248C STK11 D194V CDKN2A W110* FGFR3 S249C MET Y1248H STK11 E199* CSF1R L301* FGFR3 S371C MLH1 V384D STK11 E199K CSF1R L301S FGFR3 Y373C MYC A59V STK11 E57fs*7 CSF1R Y969* FLT3 D835del MYC N101T STK11 F264fs*22 CSF1R Y969* FLT3 D835E MYC P260A STK11 G196V CSF1R Y969C FLT3 D835E MYC P57S STK11 P281fs*6 CSF1R Y969F FLT3 D835H MYC S77F STK11 P281L CSF1R Y969H FLT3 D835V MYC T73I STK11 Q170* CTNNB1 A13T FLT3 D835Y NRAS A18T STK11 Q37* CTNNB1 A21T FLT3 D835Y NRAS G12A STK11 W332* CTNNB1 D32A FLT3 I836del NRAS G12C TP53 G245S CTNNB1 D32G FLT3 I836M NRAS G12C TP53 R175H

9 CTNNB1 D32H FLT3 Y572C NRAS G12D TP53 R248Q CTNNB1 D32N HRAS G12C NRAS G12D TP53 R248W CTNNB1 D32V HRAS G12D NRAS G12R TP53 R273C CTNNB1 D32Y HRAS G12R NRAS G12S TP53 R273H CTNNB1 D32Y HRAS G12V NRAS G12S TP53 R306* CTNNB1 G34E HRAS G12V NRAS G12V VHL F148fs*11 CTNNB1 G34R HRAS G13C NRAS G13A VHL L158Q CTNNB1 G34V HRAS G13C NRAS G13C VHL L85P CTNNB1 G34V HRAS G13R NRAS G13C VHL L89H CTNNB1 S33C HRAS G13S NRAS G13D VHL P81S CTNNB1 S33F HRAS Q61H NRAS G13D VHL R161* CTNNB1 S33F HRAS Q61H NRAS G13R VHL R167W *460 Assays (396 unique somatic mutations; 460 count includes bidirectional assay sets)

10 Table S7. OncoMap 3 Extended Assays* GENE_SYMBOL AA_CHANGE GENE_SYMBOL AA_CHANGE GENE_SYMBOL AA_CHANGE GENE_SYMBOL AA_CHANGE ABL1 E459K EPHA4 G370E MAP2K4 N234I PIK3R1 W583del ABL1 F311L EPHA4 S399F MAP2K4 Q142L PKHD1 E1806K ABL1 F486S EPHA4 T893T MAP2K4 R154W PKHD1 L1096R ABL1 H396P EPHA5 E503K MAP2K4 R274G PKHD1 R19H ABL1 S417Y EPHA5 G582E MAP2K4 R304* PTCH1 M457fs*34 ABL1 T315I EPHA5 N1032S MAP2K4 S184L PTCH1 M561R ABL1 T315N EPHA5 R417Q MAP2K4 S251N PTCH1 Q365* ABL2 E63Q EPHA5 T856I MAP2K4 S280* PTCH1 Q417* ADAMTSL3 A1315E EPHA5 Y796H MAP2K4 W310* PTCH1 R770* ADAMTSL3 R855C EPHA8 R179C MEN1 A368fs*7 PTEN F347fs*13 ADAMTSL3 R855H EPHB1 E698* MEN1 G208fs*16 PTEN G129R ADAMTSL3 V330M EPHB1 G499C MEN1 I125fs*54 PTEN H61R ALK A877S EPHB1 I719V MEN1 R98* PTEN I101T ALK L560F EPHB1 N753N MET E168D PTEN Q171* ALK P496L EPHB1 P420fs*28 MET L1213V PTEN Q214* ALK P542R EPHB1 R390L MET N1118Y PTEN R130L ALK R1275Q EPHB1 R56P MET Y1253D PTEN T319fs ALK R401* EPHB1 R743Q MLL3 C347G PTEN V290fs*1 APC R2166* EPHB1 S670C MLL3 D400N PTEN Y68H APC S1465fs*3 EPHB6 A588P MLL3 L478W PTEN Y76fs ATM P604S EPHB6 D345N MLL3 R1906X PTPN11 A72D ATM R3008C EPHB6 D915G MLL3 T3698S PTPN11 A72T ATM R3008H EPHB6 E860K MPL S204F PTPN11 A72V AURKA S155R EPHB6 P728S MPL W515L PTPN11 D61V AURKA S361* EPHB6 R704Q MSH2 E467K PTPN11 D61Y AURKA V174M ERBB2 A775_G776insYVMA MSH2 L787fs*11 PTPN11 E69K AURKB L21L ERBB2 C334S MSH6 K247fs*32 PTPN11 E76A AURKC E114Q ERBB2 D326G MSH6 P1087fs*3 PTPN11 E76G AURKC G18E ERBB2 E321G MSH6 P1087fs*5 PTPN11 E76K AURKC H210Q ERBB2 G776V MYC E54D PTPN11 E76Q AXL R295W ERBB2 I861fs MYC P72T PTPN11 E76V BMX R670W ERBB2 N319D MYC P74A PTPN11 G503A BRAF G596D ERBB2 T216S MYC P74W PTPN11 G60V BRAF L618S ERBB2 V750E MYC P75A PTPN11 T73I BRAF P453T ERBB2 V777A MYC P75H RAF1 Q335H BRAF R462I FBXW7 R224* MYC P78L RAF1 S259A BRAF S605G FBXW7 R278* MYC P78L RB1 Q685* BRAF S614P FBXW7 R393* MYC P78S RB1 Y606* BRAF V600_K601>E FBXW7 R465C MYC Q52E RET A664D BRAF V600E FBXW7 R465H MYC Q52H RET A876V BRCA1 L30F FBXW7 R479Q MYC S107N RET A919V BRCA1 S114T FBXW7 R505C MYC S107T RET C609Y BRCA1 V757fs*8 FES M704V MYC T73I RET C618Y BRCA2 E1593* FES R706Q MYH1 A1445T RET C630R BRCA2 K1517fs*23 FES S759F MYH1 S1306L RET D925H BRCA2 N1784fs*7 FES V743M MYH1 V1598M RET E632_L633del CDH1 D370H FGFR2 D101Y NF1 A422P RET E884K CDH1 I374I FGFR2 K659E NF1 H2637Y RET E901K CDH1 T263fs*3 FGFR2 K659M NF1 K1444E RET F612_C620del CDH1 T399fs*17 FGFR2 K659N NF1 R1534* RET G748C CDK4 R24C FGFR2 K659R NF1 Y1607fs*17 RET M918T CDKN2A D84V FGFR2 P253R NF2 Q212* RET P766S CDKN2A L78fs*41 FGFR2 Y375C NF2 Q337* RET R908K CDKN2A P48L FGFR3 A391E NF2 Q400* ROBO1 P1445R CDKN2A S43I FGFR3 E322K NF2 Q410* ROBO1 V292L CEBPA G36fs*124 FGFR3 F384L NF2 R196* ROBO2 G1339* CEBPA K313del FGFR3 H284fs*10 NF2 R262* ROBO2 S819N CEBPA N307_V308insQRN FGFR3 K650E NF2 R341* ROBO2 T48K CEBPA Q83fs*77 FGFR3 K650M NF2 R466* ROBO2 W870R CEBPA S21fs*139 FGFR3 K650T NF2 R57* ROS1 F2138S CREBBP N83T FGFR3 Y241C NF2 V219M ROS1 Q865H CREBBP P937S FGFR4 P672T NOTCH1 A1701P ROS1 Y419H CSF1R L301V FGFR4 S732N NOTCH1 I1680N RUNX1 D171G CSF1R Y969D FGFR4 V510M NOTCH1 L1574P RUNX1 D171N CSF1R Y969N FLNB A1534G NOTCH1 L1585P RUNX1 R135G CTNNB1 A43T FLNB N663K NOTCH1 L1593P RUNX1 R139* CTNNB1 D58N FLNB R566Q NOTCH1 L1600P RUNX1 R139G CTNNB1 E53K FLNB T703K NOTCH1 L1678P RUNX1 R174Q CTNNB1 G38P FLT3 A627T NOTCH1 P2514fs*4 RUNX1 R177Q CTNNB1 H36P FLT3 A680V NOTCH1 Q2459* RUNX1 R80C CTNNB1 H36Y FLT3 D835N NOTCH1 R1598P SIX4 D780E CTNNB1 I35S FLT3 G64G NOTCH1 V1578del SIX4 G446D CTNNB1 K49R FLT3 L561L NPM1 W288fs*12 SMAD2 D300V CTNNB1 N287S FLT3 R834_D835del NPM1 W290fs*10 SMAD4 D351H CTNNB1 P44S FLT3 S451F NRAS A11T SMAD4 D351N CTNNB1 S29F FLT3 V592G NRAS A59T SMAD4 G386R CTNNB1 S33A FLT4 Y1333H NRAS G12G SMAD4 P130S CTNNB1 S33L FSCB Q764K NRAS G12P SMAD4 Q245* CTNNB1 S33P FSCB S775L NRAS G13G SMAD4 R361H CTNNB1 S45del FSCB T262R NRAS Q61Q SMAD4 R445* CTNNB1 S47N FYN G410R NRAS S65C SMAD4 R515* CTNNB1 T40I FYN V243L NRAS Y64N SMAD4 S343* CTNNB1 T41A FYN W290* NTRK1 A107V SMAD4 V350D CTNNB1 T41S GATA1 A53fs*7 NTRK1 L213L SMARCB1 A382fs*4 CTNNB1 T42I GATA1 P50fs*87 NTRK2 L138F SMARCB1 E216* CTNNB1 W25_D32del GATA1 Q17* NTRK3 L336Q SMARCB1 G317fs*3 CUBN A2252V GATA1 S30fs*9 NTRK3 R678Q SMARCB1 P383fs*3 CUBN H786Q GATA1 V74I NTRK3 R721F SMARCB1 R158* CUBN I3189V GNAS E110K NTRK3 T149R SMARCB1 R201* DBN1 E278K GNAS Q227L NTRK3 V307L SMARCB1 R377H DBN1 E640Q GNAS R201C PAK7 T397K SMARCB1 R40* DDR1 A497S GNAS R201H PALB2 D1168G SMARCB1 Y47* DDR1 F823_R824>FW GUCY1A2 E681V PALB2 P1008L SMO R199W DDR2 R105S GUCY1A2 N685T PDGFRA D1071N SMO T640A EGFR A289T GUCY1A2 Q119* PDGFRA D842_D846>S SPTAN1 D1918N EGFR A289V HNF1A W206C PDGFRA D842V SPTAN1 P1017S EGFR A597P HNF1A W206L PDGFRA F808L SPTAN1 R1794W EGFR C620Y HRAS G12S PDGFRA G829R SPTAN1 S904C EGFR E709A HRAS G13D PDGFRA N659K STK11 Q214* EGFR E709G HRAS G13V PDGFRA N659Y SUFU F197F EGFR E709K IGF1R A1347V PDGFRA N870S SUFU P187L EGFR E746_A750>IP IGF1R Q1214* PDGFRA R560_S564del SUFU S377fs*7 C361 EGFR E746_A750del IGF1R R895W PDGFRA T674I TBX22 A51T

11 GENE_SYMBOL AA_CHANGE GENE_SYMBOL AA_CHANGE GENE_SYMBOL AA_CHANGE GENE_SYMBOL AA_CHANGE EGFR E746_S752>V IGF1R S1278_F1279>F PDGFRA V824V TBX22 D307N EGFR H835L JAK2 K539L PDGFRB P1090P TBX22 V16A EGFR K745_E749del JAK2 T875N PDGFRB T882I TEC R563K EGFR L62R JAK3 L527P PDGFRB Y589H TFDP1 R168H EGFR L730F KIT D579del PDPK1 D527E TIAM1 H847Y EGFR L747_T751>P KIT D816F PDPK1 T354M TIAM1 S1148I EGFR L747_T751del KIT D820G PIK3CA *1069W TP53 A161T EGFR L833V KIT K550_V555del PIK3CA C901F TP53 C135F EGFR L858L KIT K558>NP PIK3CA E1034G TP53 E285K EGFR L858R KIT V559_E561del PIK3CA E453K TP53 G245C EGFR L861Q KIT V559_V560del PIK3CA E542V TP53 G266* EGFR R108K KIT V560del PIK3CA E545D TP53 P309S EGFR R222C KIT V825I PIK3CA G1007R TP53 R213* EGFR R252C KIT W557_E561del PIK3CA G1049A TP53 R273L EGFR R677H KIT W557_K558del PIK3CA G1049R TP53 R282W EGFR R680G KIT W557_K558del PIK3CA G1049S TP53 R342* EGFR S703F KIT W557_V559>C PIK3CA G118D TP53 T155P EGFR S752_I759del KIT W557_V559>F PIK3CA H1065L TP53 V157F EGFR S752_I759del KIT Y418_D419>G PIK3CA H556Q TP53 V272M EGFR T263P KIT Y553_Q556del PIK3CA H701P TP53 Y163C EGFR T751I KIT Y823D PIK3CA K111N TP53 Y220C EGFR V769_D770insASV KRAS A11V PIK3CA L10_M16del TRIM24 I320T EGFR V774M KRAS A146V PIK3CA M1043I TRIM24 S923S EGFR V819V KRAS A59T PIK3CA M1043V TRIM24 T403N EPHA1 A212T KRAS G12F PIK3CA P17del TRIM33 E811K EPHA1 E703K KRAS L19F PIK3CA Q546K TRIM33 M580I EPHA3 D446Y KRAS V14I PIK3CA Q546P TRIM33 P885S EPHA3 D806N LRP1B D3049Y PIK3CA Q546R TRIM33 T1085T EPHA3 G187R LRP1B D4395Y PIK3CA R1023Q TSC1 S444* EPHA3 G518L LRP1B G304R PIK3CA T1025A TSC1 T690_R692del,insNL* EPHA3 G766E LRP1B G418C PIK3CA T1025N TSHR A623S EPHA3 I621L LRP1B K1574I PIK3CA T1025S TSHR D633Y EPHA3 M269I LRP1B L1172V PIK3CA T1052K TSHR I630L EPHA3 N379K LRP1B M3709I PIK3CA Y1021H TSHR T632I EPHA3 N85S LRP1B N2366K PIK3R1 D260Y VHL E160K EPHA3 R728L LRP1B P1337S PIK3R1 DKRMNS560del VHL G114C EPHA3 S229F LRP1B R1987L PIK3R1 E439del VHL I151S EPHA3 S229Y LRP1B R4020K PIK3R1 G76R VHL Q96* EPHA3 S449F LRP1B S2834F PIK3R1 I259_N264del VHL S65L EPHA3 T166N LYN D385Y PIK3R1 K459delN VHL S68* EPHA3 T37K MAP2K4 A279T PIK3R1 N264K VHL S80R EPHA3 T660K MAP2K4 E221* PIK3R1 N564K VHL V130L EPHA3 W250R MAP2K4 I295fs*23 PIK3R1 R574fs WT1 R458* EPHA4 D867N MAP2K4 K309N PIK3R1 T576del *587 assays covering 110 genes

12 Figure S1. Correlation between BRAF V600E abundance and Langerhans cell proportion in LCH cases For each patient sample in which mutant BRAF alleles were detected by OncoMap hme analysis, the proportion of mutant alleles was determined as described in Supplemental Methods and correlated with the proportion of Langerhans cells in the same sample (r=0.68, p<0.0001).

13 Figure S2. Immunohistochemical analysis of BRAF pathway activation in LCH (A) A typical LCH case stained with hematoxylin and eosin shows numerous histiocytes with characteristic folded nuclei accompanied by neutrophils and eosinophils (arrowhead). (B) The identification of these cells as Langerhans cells is confirmed by their uniformly positive staining for CD1a. (C, D) BRAF pathway activation is indicated by strong cytoplasmic staining for phosphomek (C) and nuclear staining for phospho-erk (D).

14 Figure S3. Correlation between BRAF mutant allele frequency in each sample determined by OncoMap and pyrosequencing Scatter plot comparing the proportion of BRAF V600E alleles for each sample in which these proportions were determined both by OncoMap and pyrosequencing (r=0.75, p<0.0001).