Molecular Profiling of Multiple Primary Merkel Cell Carcinoma to Distinguish Genetically Distinct Tumors from Clonally Related Metastases

Supplementary Online Content

Harms KL, Lazo de la Vega L, Hovelson DH, et al. Molecular profiling of multiple primary merkel cell carcinoma to distinguish genetically distinct tumors from clonally related metastases. JAMA Dermatol. Published online April 12, 2017. doi:10.1001/jamadermatol.2017.0507

eMethods. eFigure 1. Clinically designated multiple MCC primaries display similar Merkel cell polyomavirus status eFigure 2. Mutation (variant) allele frequency in Merkel cell carcinoma tumors eTable 1. Library Quality Parameters eTable 2. A, Primer pairs used for Merkel cell polyomavirus PCR; B, Merkel Cell Polyomavirus PCR-Sanger results eTable 3. Somatic Mutations Detected in Merkel Cell Carcinoma Cases eTable 4. Copy Number Alterations in Merkel Cell Carcinoma Cases eTable 5. Primary-metastasis pairs used for similarity index comparison eTable 6. Tumor purity estimates based on hematoxylin and eosin stained sections eReferences.

This supplementary material has been provided by the authors to give readers additional information about their work.

Downloaded From: https://jamanetwork.com/ on 09/27/2021 eMethods

Cohort

MCC cases that were clinically designated as multiple primary tumors were identified from a database of 473 cases compiled by the Multidisciplinary MCC Program. All cases were discussed at the Multidisciplinary MCC tumor board for consensus treatment recommendations. For a clinical designation of distinct primary tumors, MCC tumors were either spatially and/or temporally separated such that a cutaneous local recurrence or in-transit metastasis was not suspected. Re-staging with imaging studies at the time of the second primary tumor was negative for distant stage IV disease.

Targeted Next Generation Sequencing

Targeted NGS assessing the complete coding sequence of 409 cancer related genes on archived FFPE (formalin-fixed paraffin embedded) material to identify somatic mutations and CNAs was performed using the Ion Ampliseq Comprehensive Cancer Panel (CCP) as described previously 1,2. Briefly, DNA was isolated from macrodissected FFPE sections using the Qiagen Allprep FFPE DNA/RNA kit (Qiagen, Valencia, CA). DNA was quantified using the Qubit 2.0 fluorometer (Life Technologies, Foster City, CA). Barcoded libraries were generated using 40ng of DNA amplified using the Ion AmpliSeq Library Kit 2.0 (Life Technologies, Foster City, CA) to perform targeted, multiplexed PCR based next generation sequencing (NGS) using the Ion Ampliseq Comprehensive Cancer Panel (CCP), which targets 1,688,650 bases from 15,992 amplicons representing 409 cancer genes (http://tools.invitrogen.com/downloads/cms_103573.csv). Templates were prepared using the Ion PI-HiQ Template OT2 200 Kit (Life Technologies, Foster City, CA) on the Ion One Touch 2 according to the manufacturer’s instructions. NGS of multiplexed templates was performed on Ion Proton P1 chips using the Ion P-HiQ Sequencing 200 Kit according to the manufacturer’s instructions.

Somatic Variant Identification

NGS data analysis was performed essentially as described 1,2 using in house pipelines based on Torrent Suite 4.0 and Torrent Mapping Alignment Program using default parameters. Single nucleotide variants (SNVs) and insertions/deletions (indels) were identified using the Torrent Variant Caller plugin using default low-stringency somatic variant settings. Called variants were filtered to remove non-coding variants, those with flow corrected read depths (FDP) ≤50, flow corrected variant allele containing reads (FAO) ≤6, variant allele frequencies (FAO/FDP) <0.10, extreme skewing of forward/reverse flow corrected reads (FSAF/FSAR <0.2 or >5), or indels within homopolymer runs >4 bases. Variants occurring exclusively in reads with other single nucleotide variants or indels and those occurring in the last mapped base of a read were excluded. Additionally, any variants called in >4% of internally sequenced samples using the same panel and not reported in COSMIC were removed. Nonsynonymous variants seen in more than 6 samples sequenced on the same panel and not observed in COSMIC were also excluded. Variants present in ESP6500 or 1000 Genomes (from Annovar) as well as variants present in the ExAC database (http://exac.broadinstitute.org) that are not at known COSMIC hotspots were considered germ line variants and removed. All high confidence variants passing the above criteria were then visualized in IGV. We have previously demonstrated that our filtering criteria identify variants that pass Sanger sequencing validation with >95% accuracy. From these somatic variants, hotspots (>1 observation at that residue in COSMIC) in oncogenes, or hotspot or deleterious alterations (nonsense/frameshift variants) in tumor suppressors were then considered as prioritized variants.

Downloaded From: https://jamanetwork.com/ on 09/27/2021 Copy Number Analysis

To identify copy number alterations (CNAs), for each sample, normalized GC content corrected read counts per amplicon were divided by those from a composite “normal” male DNA sample generated from multiple individual and pooled normal FFPE tissues, yielding a copy number ratio for each amplicon as described 1-3. Gene-level copy number estimates were determined by taking the coverage-weighted mean of the per-probe ratios, with expected error determined by the probe-to-probe variance. Library quality parameters are described in Supplemental Table S5.

Clonality Analysis

Similarity index was calculated as previously described 4 using the equation:

Similarity Index = NS / (NS + NO + NU)

Where NS = number of shared changes, NO = number of opposite changes, and NU = number of unique changes. Shared changes were counted once between cases (e.g. the DST c.A3835G mutation identified in both MCC28 and MCC29 was counted as a single shared change for the similarity index calculation). No events were in the “opposite changes” category in our cases. For mutational analysis, variant calls were considered to be shared events if they demonstrated the same nucleotide change at the same nucleotide position in a given gene.

MCPyV PCR

Detection of MCPyV sequences in tumor DNA was performed by qPCR as previously described 5,6. Primer/probe sets were LT2 (large T antigen) and SET9 (small T antigen), with cell line controls including MKL2 (MCC, positive control) and A375 (melanoma, negative control).

MCPyV genomic sequencing was performed by PCR-Sanger using previously reported primers 7 as well as custom primers targeting the 5’ region of LTAg exon 2 and optimized for FFPE (Supplemental Table S2). Sanger sequencing was performed at the University of Michigan DNA Sequencing Core as previously described 1.

Downloaded From: https://jamanetwork.com/ on 09/27/2021 eFigure 1. Clinically-designated multiple MCC primaries display similar Merkel cell polyomavirus status

Quantitative PCR was performed on tumor DNA to detect genomically integrated Merkel cell polyomavirus (MCPyV) sequences. Primer pairs targeted viral small T antigen (SET9) sequences. MKL2: positive control Merkel cell carcinoma cell line. A375: negative control melanoma cell line. sTAG: small T antigen.

Downloaded From: https://jamanetwork.com/ on 09/27/2021 eFigure 2. Mutation (variant) allele frequency in Merkel cell carcinoma tumors

(a) Allele frequency per mutational event per sample demonstrates lower variant allele frequency in MCPyV-positive samples (MCC23-29) compared to MCPyV-negative samples (MCC30-32). (b) Mutations observed in MCPyV-positive tumors in this cohort displayed significantly lower variant allele frequency (Student’s t-test, p < 0.001). (c) Variant allele frequency was compared in an independent cohort of previously sequenced Merkel cell carcinoma tumors (Harms et al, 2015), including 5255 mutational events across 8 Merkel cell polyomavirus (MCPyV)-negative tumors and 214 mutational events across 7 MCPyV-positive tumors. Allele frequency was significantly lower in MCPyV-positive tumors (Student’s t- test, p < 0.001). (d) Proposed model for generation of non-overlapping, low allele frequency mutations in MCPyV-positive MCC tumors, as compared to clonal driver mutations in MCPyV-negative tumors.

Downloaded From: https://jamanetwork.com/ on 09/27/2021 eTable 1. Library Quality Parameters

% Synonymous Nonsynonymous Total Mapped Mean Reads Calls Variants Prioritized Sample Uniformity Called 4 Reads Depth on 1 Passing Passing Variants Variants 2 3 Target Filtering Filtering MCC23 4,406,584 278.4 87.61% 99.25% 2078 2 2 0 MCC24 5,434,999 335 89.59% 98.12% 9930 37 30 1 MCC25 10,838,606 687.6 90.27% 99.23% 1247 0 0 0 MCC26 7,161,117 440.6 86.42% 98.50% 4559 13 17 1 MCC28 12,014,696 777.7 89.57% 99.40% 1107 1 1 0 MCC29 13,896,390 891.9 88.45% 99.48% 1081 1 3 0 MCC30 8,673,172 558.7 90.64% 99.19% 1252 19 41 12 MCC31 10,928,287 702.8 84.29% 99.30% 1209 20 48 8 MCC32 11,659,542 755.4 87.65% 99.50% 1209 19 45 9 Average 9,445,933 603.1 88.28% 99.11% 2,630 12 21 3 Sequencing statistics for 9 Merkel Cell Carcinomas profiled by next generation sequencing (NGS) using the Ion Ampliseq Comprehensive Cancer Panel (CCP), which targets 1,688,650 bases in 15,992 amplicons representing 409 cancer genes. Given are the number of mapped reads per sample, the mean read coverage depth over targeted bases, uniformity of mapped reads and the % reads on target, as well as the average called variants prior to any filtering. 1Variants called by automated low stringency variant calling. 2Synonymous variants passing stringent filtering. 3Variants passing filtering of technical artifacts, poorly supported variants, germline SNPs and synonymous/non-coding variants. 4Variants passing filtering described in 2 and prioritized as likely driving oncogenic or tumor suppressive mutations.

Downloaded From: https://jamanetwork.com/ on 09/27/2021 eTable 2A. Primer pairs used for Merkel cell polyomavirus PCR

Primer pair Forward Reverse Probe Reference FAM- LT2 CCCGATATACCTCCCGAACAC CTGGTTCCATTGGGTGTGCTG TCTTCCTGAATTGGTGGTCTCCTCTCTGCT- Rodig et al BHQ

SET9 TTAGCTGTAAGTTGTCTCGCC CACCAGTCAAAACTTTCCCAAG FAM-AAACACTCTCCCCACGTCAGACAG-BHQ Rodig et al

AGTEX2-1 TACTGCTTACTGCATCTGCACC GGCGAGCTTCTTGAGGAG NA Laude et al

AGTEX2-2 GCGATGAATCACTTTCCTCC CCAATTACAGCTGGCCTCTT NA Laude et al

AGTEX2-3 CCCCTTACAAATTACTGCAAGAG GATGGACAGTTTATATTCAAGGCC NA Laude et al

AGTEX2-4 GCTTTGCTGCAGCCTTAATAG CAAACACAGGAAATATGAAGCAG NA Laude et al

AGTEX2A TGTAAAACGACGGCCAGTcccaatggaaccagtgtaccta CAGGAAACAGCTATGACCgagaatggaggaggggtcttc NA new

AGTEX2B TGTAAAACGACGGCCAGTgaggcctcgtcatctcagtt CAGGAAACAGCTATGACCaaatggcaaaacaacttactgtt NA new

LT2 and SET9 primer/probes were used for Merkel cell polyomavirus copy number analysis. Other primer pairs were used for PCR-Sanger analysis.

Downloaded From: https://jamanetwork.com/ on 09/27/2021 eTable 2B. Merkel Cell Polyomavirus PCR-Sanger results

PCR results by primer pair AGTEX2- AGTEX2- AGTEX2- AGTEX2- Sample LT2 SET9 1 2 3 4 AGTEX2A AGTEX2B MCC23 + + ND ND ND ND + + MCC24 + + ------MCC25 + + + + + + + + MCC26 + + - - - - + + MCC28 + + ND ND ND ND ND + MCC29 + + ND ND ND ND ND + MCC30 - - ND ND ND ND ND ND MCC31 - - ND ND ND ND ND ND MCC32 - - ND ND ND ND ND ND

Sanger results %Homology Reference Sample Sample Coverage (# bp) NC_010277.1 EurCauC1 MCC23 MCC25 MCC26 MCC28 MCC29 MCC23 394 98 100 100 100 100 100 100 MCC24 N/A N/A N/A N/A N/A N/A N/A N/A MCC25 2071 99 99 100 100 100 100 100 MCC26 394 98 100 100 100 100 100 100 MCC28 237 97.4 100 100 100 100 100 100 MCC29 233 97.4 100 100 100 100 100 100 MCC30 N/A N/A N/A N/A N/A N/A N/A N/A MCC31 N/A N/A N/A N/A N/A N/A N/A N/A MCC32 N/A N/A N/A N/A N/A N/A N/A N/A

ND: not done due to insufficient remaining DNA. N/A: not applicable (no amplicon for analysis).

Downloaded From: https://jamanetwork.com/ on 09/27/2021 eTable 3. Somatic Mutations Detected in Merkel Cell Carcinoma Cases

Var. Desig- Sam- AA Allele Cosmic Pt MCV nation ple Location Gene REF ALT Change Freq. Transcript Nuc change ID Paired or Nonpaired COSM168 4 Negative Primary MCC32 chr1:11184573 MTOR G A p.S2215F 0.415 NM_004958 c.C6644T Pair 31/32 6998 1 Positive Primary MCC23 chr7:148524300 EZH2 G C p.S228S 0.479 NM_004456 c.C684G NA Pair 23/24 COSM168 4 Negative Metastasis MCC31 chr1:11184573 MTOR G A p.S2215F 0.49 NM_004958 c.C6644T Pair 31/32 6998 1 Positive Primary MCC24 chr7:148524300 EZH2 G C p.S228S 0.462 NM_004456 c.C684G NA Pair 23/24 1 Positive Primary MCC24 chrX:48652530 GATA1 C T p.P401S 0.294 NM_002049 c.C1201T NA nonpaired 1 Positive Primary MCC24 chr8:145742800 RECQL4 C T p.E71K 0.196 NM_004260 c.G211A NA nonpaired 1 Positive Primary MCC24 chr17:75484830 Sept9 C T p.F364F 0.175 NM_006640 c.C1092T NA nonpaired 4 Negative Primary MCC32 chr1:11270952 MTOR G A p.F1191F 0.452 NM_004958 c.C3573T NA Pair 31/32 4 Negative Metastasis MCC31 chr1:11270952 MTOR G A p.F1191F 0.484 NM_004958 c.C3573T NA Pair 31/32 4 Negative Primary MCC32 chr1:114948307 TRIM33 C G p.L831F 0.494 NM_015906 c.G2493C NA Pair 31/32 1 Positive Primary MCC24 chrX:100611237 BTK G A p.L457L 0.164 NM_000061 c.C1369T NA nonpaired 1 Positive Primary MCC24 chrX:41043740 USP9X C T p.P1124S 0.164 NM_001039590 c.C3370T NA nonpaired 4 Negative Metastasis MCC31 chr1:114948307 TRIM33 C G p.L831F 0.525 NM_015906 c.G2493C NA Pair 31/32 4 Negative Primary MCC32 chr1:120464931 NOTCH2 CC TT p.G1714N 0.481 NM_024408 c.5140_5141AA NA Pair 31/32 4 Negative Metastasis MCC31 chr1:120464931 NOTCH2 CC TT p.G1714N 0.5 NM_024408 c.5140_5141AA NA Pair 31/32 1 Positive Primary MCC24 chr6:117662360 ROS1 G A p.Q1673X 0.159 NM_002944 c.C5017T NA nonpaired PDE4DI 4 Negative Primary MCC30 chr1:144922211 G A p.H319H 0.478 NM_014644 c.C957T NA nonpaired P 4 Negative Primary MCC32 chr1:145534123 ITGA10 CC TT p.P543L 0.43 NM_003637 c.1628_1629TT NA Pair 31/32 1 Positive Primary MCC24 chr6:152680575 SYNE1 G A p.L3447L 0.157 NM_033071 c.C10339T NA nonpaired 1 Positive Primary MCC24 chrX:48652388 GATA1 G A p.L353L 0.157 NM_002049 c.G1059A NA nonpaired 4 Negative Metastasis MCC31 chr1:145534123 ITGA10 CC TT p.P543L 0.47 NM_003637 c.1628_1629TT NA Pair 31/32 1 Positive Primary MCC24 chr2:5833294 SOX11 C T p.G147G 0.154 NM_003108 c.C441T NA nonpaired 4 Negative Primary MCC32 chr1:145539751 ITGA10 C T p.V1061V 0.418 NM_003637 c.C3183T NA Pair 31/32 1 Positive Primary MCC24 chrX:41075551 USP9X G A p.D1911N 0.153 NM_001039590 c.G5731A NA nonpaired COSM183 1 Positive Primary MCC24 chr3:10183639 VHL G A p.E36E 0.15 NM_000551 c.G108A nonpaired 60 4 Negative Metastasis MCC31 chr1:145539751 ITGA10 C T p.V1061V 0.496 NM_003637 c.C3183T NA Pair 31/32 4 Negative Primary MCC30 chr1:156841435 NTRK1 C T p.S246S 0.458 NM_002529 c.C738T NA nonpaired 1 Positive Primary MCC24 chr1:147095823 BCL9 C T p.S1115L 0.147 NM_004326 c.C3344T NA nonpaired 1 Positive Primary MCC24 chr9:139400235 NOTCH1 G A p.T1371T 0.145 NM_017617 c.C4113T NA nonpaired 4 Negative Primary MCC30 chr1:156849880 NTRK1 C T p.S712S 0.427 NM_002529 c.C2136T NA nonpaired 4 Negative Primary MCC30 chr1:179079460 ABL2 CC TT p.G594E 0.467 NM_005158 c.1736_1737AA NA nonpaired TNFRSF 4 Negative Primary MCC32 chr1:2494663 C T p.A268V 0.454 NM_003820 c.C803T NA Pair 31/32 14

Downloaded From: https://jamanetwork.com/ on 09/27/2021 1 Positive Primary MCC24 chr9:139400306 NOTCH1 C T p.D1348N 0.145 NM_017617 c.G4042A NA nonpaired TNFRSF 4 Negative Metastasis MCC31 chr1:2494663 C T p.A268V 0.491 NM_003820 c.C803T NA Pair 31/32 14 4 Negative Metastasis MCC31 chr1:27057928 ARID1A C T p.Q546X 0.457 NM_006015 c.C1636T NA Pair 31/32 1 Positive Primary MCC24 chrX:79281119 TBX22 C T p.S159L 0.145 NM_016954 c.C476T NA nonpaired 2 Positive Primary MCC26 chr19:45260308 BCL3 C T p.P185L 0.11 NM_005178 c.C554T NA nonpaired 1 Positive Primary MCC24 chr4:41750474 PHOX2B C T p.G52R 0.143 NM_003924 c.G154A NA nonpaired 1 Positive Primary MCC24 chr16:15841924 MYH11 G A p.V720V 0.14 NM_002474 c.C2160T NA nonpaired 4 Negative Primary MCC32 chr1:27057928 ARID1A C T p.Q546X 0.49 NM_006015 c.C1636T NA Pair 31/32 4 Negative Primary MCC32 chr1:27099853 ARID1A C T p.P1244P 0.412 NM_006015 c.C3732T NA Pair 31/32 4 Negative Metastasis MCC31 chr1:27099853 ARID1A C T p.P1244P 0.459 NM_006015 c.C3732T NA Pair 31/32 4 Negative Primary MCC32 chr1:32745693 LCK C T p.P403P 0.473 NM_005356 c.C1209T NA Pair 31/32 4 Negative Metastasis MCC31 chr1:32745693 LCK C T p.P403P 0.513 NM_005356 c.C1209T NA Pair 31/32 4 Negative Primary MCC30 chr1:9786967 PIK3CD G A p.D1000N 0.613 NM_005026 c.G2998A NA nonpaired chr11:10817240 4 Negative Primary MCC30 ATM C T p.T1735T 0.467 NM_000051 c.C5205T NA nonpaired 2 chr11:10821655 4 Negative Primary MCC30 ATM C A p.Y2833X 0.455 NM_000051 c.C8499A NA nonpaired 0 chr11:12835032 4 Negative Primary MCC32 ETS1 G A p.S251F 0.473 NM_005238 c.C752T NA Pair 31/32 5 chr11:12835032 4 Negative Metastasis MCC31 ETS1 G A p.S251F 0.442 NM_005238 c.C752T NA Pair 31/32 5 chr12:13255409 4 Negative Primary MCC32 EP400 C T p.A2976V 0.47 NM_015409 c.C8927T NA Pair 31/32 2 chr12:13255409 4 Negative Metastasis MCC31 EP400 C T p.A2976V 0.514 NM_015409 c.C8927T NA Pair 31/32 2 ADAMTS 4 Negative Primary MCC30 chr12:43858419 GG AA p.P495F 0.569 NM_025003 c.1483_1484TT NA nonpaired 20 4 Negative Primary MCC30 chr12:46245005 ARID2 C T p.P1033P 0.497 NM_152641 c.C3099T NA nonpaired 1 Positive Primary MCC24 chr6:41565644 FOXP4 C T p.S604S 0.137 NM_138457 c.C1812T NA nonpaired 1 Positive Primary MCC24 chr9:98209403 PTCH1 C T p.V1379I 0.136 NM_000264 c.G4135A NA nonpaired 4 Negative Metastasis MCC31 chr12:46245783 ARID2 C T p.L1293F 0.626 NM_152641 c.C3877T NA Pair 31/32 4 Negative Primary MCC32 chr12:46245783 ARID2 C T p.L1293F 0.386 NM_152641 c.C3877T NA Pair 31/32 4 Negative Primary MCC32 chr12:49425885 KMT2D G A p.L4201L 0.438 NM_003482 c.C12603T NA Pair 31/32 4 Negative Metastasis MCC31 chr12:49425885 KMT2D G A p.L4201L 0.484 NM_003482 c.C12603T NA Pair 31/32 4 Negative Primary MCC32 chr12:49440062 KMT2D G A p.Q1522X 0.486 NM_003482 c.C4564T NA nonpaired 1 Positive Primary MCC24 chr9:139399826 NOTCH1 G A p.Q1508X 0.135 NM_017617 c.C4522T NA nonpaired 4 Negative Metastasis MCC31 chr12:6782552 ZNF384 GG AA p.S247F 0.484 NM_133476 c.740_741TT NA nonpaired COSM130 4 Negative Primary MCC30 chr13:48955538 RB1 C T p.R552X 0.853 NM_000321 c.C1654T 0225, nonpaired COSM887 COSM116 2722, 4 Negative Primary MCC32 chr13:49030434 RB1 C T p.Q637X 0.901 NM_000321 c.C1909T Pair 31/32 COSM116 2723

Downloaded From: https://jamanetwork.com/ on 09/27/2021 COSM116 2722, 4 Negative Metastasis MCC31 chr13:49030434 RB1 C T p.Q637X 0.946 NM_000321 c.C1909T Pair 31/32 COSM116 2723 1 Positive Primary MCC24 chr7:128845486 SMO C T p.R261R 0.133 NM_005631 c.C783T NA nonpaired 1 Positive Primary MCC24 chr19:3119318 GNA11 C T p.L284L 0.127 NM_002067 c.C850T NA nonpaired 1 Positive Primary MCC24 chr19:3119323 GNA11 C T p.Y285Y 0.127 NM_002067 c.C855T NA nonpaired 1 Positive Primary MCC24 chr17:75483505 Sept9 G A NA 0.127 NM_006640 c.860-1G>A NA nonpaired 1 Positive Primary MCC24 chr18:47800083 MBD1 C T p.D377N 0.123 NM_002384 c.G1129A NA nonpaired 4 Negative Primary MCC30 chr14:51224727 NIN C T p.E1007E 0.415 NM_020921 c.G3021A NA nonpaired p.K256N/A 4 Negative Primary MCC32 chr15:90630717 IDH2 CT TA 0.419 NM_002168 c.768_769TA NA Pair 31/32 257T 1 Positive Primary MCC24 chr7:128845574 SMO C T p.R291X 0.123 NM_005631 c.C871T NA nonpaired 1 Positive Primary MCC24 chr5:176520246 FGFR4 C T p.L389L 0.122 NM_002011 c.C1165T NA nonpaired 1 Positive Primary MCC24 chr8:38271760 FGFR1 G A p.S697F 0.12 NM_015850 c.C2090T NA nonpaired p.K256N/A 4 Negative Metastasis MCC31 chr15:90630717 IDH2 CT TA 0.551 NM_002168 c.768_769TA NA Pair 31/32 257T 4 Negative Primary MCC32 chr15:90633860 IDH2 AC TT p.V75K 0.481 NM_002168 c.223_224AA NA Pair 31/32 3 Positive Primary MCC28 chr6:152673474 SYNE1 G A p.D3741D 0.314 NM_033071 c.C11223T NA nonpaired 4 Negative Metastasis MCC31 chr15:90633860 IDH2 AC TT p.V75K 0.463 NM_002168 c.223_224AA NA Pair 31/32 4 Negative Primary MCC30 chr16:2098699 TSC2 C T p.P28L 0.497 NM_000548 c.C83T NA nonpaired 4 Negative Metastasis MCC31 chr16:3788619 CREBBP G A p.L1445L 0.496 NM_004380 c.C4335T NA nonpaired 1 Positive Primary MCC24 chr11:3697472 NUP98 G A p.P1774S 0.12 NM_016320 c.C5320T NA nonpaired 1 Positive Primary MCC24 chr7:128845507 SMO C T p.F268F 0.119 NM_005631 c.C804T NA nonpaired 1 Positive Primary MCC24 chr16:11348962 SOCS1 C T p.S125N 0.118 NM_003745 c.G374A NA nonpaired 1 Positive Primary MCC24 chr9:133760404 ABL1 G A p.K909K 0.117 NM_005157 c.G2727A NA nonpaired chr12:12143888 1 Positive Primary MCC24 HNF1A G A p.V596M 0.116 NM_000545 c.G1786A NA nonpaired 5 1 Positive Primary MCC24 chr10:8100377 GATA3 C T p.F117F 0.115 NM_002051 c.C351T NA nonpaired 1 Positive Primary MCC24 chr17:37863265 ERBB2 G A p.K32K 0.115 NM_004448 c.G96A NA nonpaired chr12:12143409 1 Positive Primary MCC24 HNF1A G A p.E329K 0.114 NM_000545 c.G985A NA nonpaired 4 4 Negative Primary MCC30 chr16:3817742 CREBBP G A p.P1077S 0.414 NM_004380 c.C3229T NA nonpaired 1 Positive Primary MCC24 chrX:79279589 TBX22 G A p.K128K 0.114 NM_016954 c.G384A NA nonpaired 1 Positive Primary MCC23 chr6:160482651 IGF2R C T p.P1127S 0.113 NM_000876 c.C3379T NA nonpaired 2 Positive Primary MCC26 chr6:41566574 FOXP4 G A p.R635K 0.124 NM_138457 c.G1904A NA nonpaired 1 Positive Primary MCC24 chrX:48546754 WAS C T p.L281L 0.112 NM_000377 c.C843T NA nonpaired 1 Positive Primary MCC24 chr6:117677821 ROS1 C T p.W1371X 0.112 NM_002944 c.G4112A NA nonpaired 1 Positive Primary MCC24 chrX:41089020 USP9X C T p.L2473L 0.111 NM_001039590 c.C7419T NA nonpaired 1 Positive Primary MCC24 chr6:32180273 NOTCH4 G A p.C886C 0.111 NM_004557 c.C2658T NA nonpaired 2 Positive Primary MCC26 chr6:41566622 FOXP4 C T p.P651L 0.163 NM_138457 c.C1952T NA nonpaired 1 Positive Primary MCC24 chr2:100170931 AFF3 C T p.G1134D 0.109 NM_002285 c.G3401A NA nonpaired 1 Positive Primary MCC24 chr7:128852266 SMO C T p.L780F 0.109 NM_005631 c.C2338T NA nonpaired

Downloaded From: https://jamanetwork.com/ on 09/27/2021 1 Positive Primary MCC24 chr6:32180287 NOTCH4 G A p.P882S 0.108 NM_004557 c.C2644T NA nonpaired 1 Positive Primary MCC24 chr19:45868165 ERCC2 G A p.Y175Y 0.107 NM_000400 c.C525T NA nonpaired 2 Positive Primary MCC26 chr16:66436616 CDH5 G A p.K633K 0.108 NM_001795 c.G1899A NA nonpaired 4 Negative Metastasis MCC31 chr16:3827645 CREBBP GG AA p.S709F 0.346 NM_004380 c.2126_2127TT NA Pair 31/32 4 Negative Primary MCC32 chr16:3827645 CREBBP GG AA p.S709F 0.344 NM_004380 c.2126_2127TT NA Pair 31/32 4 Negative Metastasis MCC31 chr16:66423403 CDH5 C A p.D253E 0.491 NM_001795 c.C759A NA Pair 31/32 2 Positive Primary MCC26 chr16:3779006 CREBBP G A p.P2014P 0.189 NM_004380 c.C6042T NA nonpaired 2 Positive Primary MCC26 chr19:45855767 ERCC2 G A p.D681D 0.114 NM_000400 c.C2043T NA nonpaired 4 Negative Primary MCC32 chr16:66423403 CDH5 C A p.D253E 0.425 NM_001795 c.C759A NA Pair 31/32 COSM664 4 Negative Primary MCC32 chr17:7576897 TP53 GG AA p.Q317X 0.905 NM_000546 c.948_949TT Pair 31/32 1 2 Positive Primary MCC26 chr16:89882291 FANCA C T p.L61L 0.1 NM_000135 c.G183A NA nonpaired COSM664 4 Negative Metastasis MCC31 chr17:7576897 TP53 GG AA p.Q317X 0.985 NM_000546 c.948_949TT Pair 31/32 1 COSM129 849, COSM107 68, COSM129 848, 4 Negative Primary MCC30 chr17:7578395 TP53 G A p.H179Y 0.874 NM_000546 c.C535T nonpaired COSM129 851, COSM129 850, COSM170 9730 4 Negative Primary MCC32 chr17:7579475 TP53 G A p.P32L 0.457 NM_001126118 c.C212T NA Pair 31/32 4 Negative Metastasis MCC31 chr17:7579475 TP53 G A p.P32L 0.541 NM_001126118 c.C212T NA Pair 31/32 2 Positive Primary MCC26 chr5:176519434 FGFR4 C T p.H280H 0.14 NM_002011 c.C840T NA nonpaired 4 Negative Primary MCC30 chr17:78320269 RNF213 C A p.P2712T 0.432 NM_001256071 c.C8134A NA nonpaired 4 Negative Primary MCC30 chr17:78350163 RNF213 C T p.S4416S 0.496 NM_001256071 c.C13248T NA nonpaired 4 Negative Primary MCC30 chr17:78359390 RNF213 G A p.W4836X 0.887 NM_001256071 c.G14508A NA nonpaired 1 Positive Primary MCC24 chr2:5833009 SOX11 G A p.P52P 0.107 NM_003108 c.G156A NA nonpaired 2 Positive Primary MCC26 chr8:37698978 GPR124 C T p.S1041F 0.107 NM_032777 c.C3122T NA nonpaired 4 Negative Primary MCC32 chr18:22805849 ZNF521 G A p.S678F 0.49 NM_015461 c.C2033T NA Pair 31/32 4 Negative Metastasis MCC31 chr18:22805849 ZNF521 G A p.S678F 0.471 NM_015461 c.C2033T NA Pair 31/32 chr13:11043590 2 Positive Primary MCC26 IRS2 G A p.I833I 0.125 NM_003749 c.C2499T NA nonpaired 2 2 Positive Primary MCC26 chr15:41804391 LTK G A p.V144V 0.113 NM_002344 c.C432T NA nonpaired 4 Negative Primary MCC30 chr18:25573486 CDH2 G A p.P379L 0.423 NM_001792 c.C1136T NA nonpaired 4 Negative Primary MCC32 chr18:47802337 MBD1 C T p.G177G 0.446 NM_002384 c.G531A NA Pair 31/32 4 Negative Metastasis MCC31 chr18:47802337 MBD1 C T p.G177G 0.491 NM_002384 c.G531A NA Pair 31/32 2 Positive Primary MCC26 chr14:36986537 NKX2-1 G A p.G354G 0.109 NM_003317 c.C1062T NA nonpaired 2 Positive Primary MCC26 chr1:19062137 PAX7 C T p.I389I 0.14 NM_002584 c.C1167T NA nonpaired

Downloaded From: https://jamanetwork.com/ on 09/27/2021 COSM138 2 Positive Primary MCC26 chr17:37829821 PGAP3 G A p.L214L 0.125 NM_033419 c.C640T nonpaired 2862 2 Positive Primary MCC26 chr10:43615064 RET C T p.Y826Y 0.125 NM_020630 c.C2478T NA nonpaired 4 Negative Primary MCC30 chr18:50976988 DCC C T p.I1116I 0.459 NM_005215 c.C3348T NA nonpaired SMARC 4 Negative Primary MCC32 chr19:11130299 C T p.P846P 0.381 NM_003072 c.C2538T NA Pair 31/32 A4 SMARC 4 Negative Metastasis MCC31 chr19:11130299 C T p.P846P 0.489 NM_003072 c.C2538T NA Pair 31/32 A4 2 Positive Primary MCC26 chr19:1622131 TCF3 G A p.P248P 0.105 NM_003200 c.C744T NA nonpaired 3 Positive Primary MCC28 chr6:152712696 SYNE1 G A p.L2581F 0.246 NM_033071 c.C7741T NA nonpaired 4 Negative Primary MCC30 chr19:17954206 JAK3 C T p.E135K 0.451 NM_000215 c.G403A NA nonpaired TNFRSF 2 Positive Primary MCC26 chr1:2489197 C T p.C34C 0.12 NM_003820 c.C102T NA nonpaired 14 3 Positive Primary MCC29 chr3:10107604 FANCD2 G T p.A776S 0.322 NM_033084 c.G2326T NA nonpaired 4 Negative Primary MCC32 chr19:18273222 PIK3R2 G A p.E339K 0.495 NM_005027 c.G1015A NA Pair 31/32 4 Negative Metastasis MCC31 chr19:18273222 PIK3R2 G A p.E339K 0.506 NM_005027 c.G1015A NA Pair 31/32 4 Negative Primary MCC32 chr19:4117620 MAP2K2 G A p.L34L 0.496 NM_030662 c.C100T NA Pair 31/32 4 Negative Metastasis MCC31 chr19:4117620 MAP2K2 G A p.L34L 0.44 NM_030662 c.C100T NA Pair 31/32 4 Negative Metastasis MCC31 chr19:42793510 CIC G T p.E438X 0.973 NM_015125 c.G1312T NA Pair 31/32 4 Negative Primary MCC32 chr19:42793510 CIC G T p.E438X 0.949 NM_015125 c.G1312T NA Pair 31/32 4 Negative Primary MCC32 chr19:42794888 CIC C T p.A656A 0.454 NM_015125 c.C1968T NA Pair 31/32 4 Negative Metastasis MCC31 chr19:42794888 CIC C T p.A656A 0.533 NM_015125 c.C1968T NA Pair 31/32 PPP2R1 4 Negative Primary MCC30 chr19:52715975 G A p.M180I 0.415 NM_014225 c.G540A NA nonpaired A 4 Negative Primary MCC30 chr2:141299395 LRP1B G A p.P2447L 0.454 NM_018557 c.C7340T NA nonpaired 4 Negative Primary MCC32 chr2:141460068 LRP1B C T p.L2026L 0.45 NM_018557 c.G6078A NA Pair 31/32 4 Negative Metastasis MCC31 chr2:141460068 LRP1B C T p.L2026L 0.507 NM_018557 c.G6078A NA Pair 31/32 COSM169 4 Negative Metastasis MCC31 chr2:141641477 LRP1B C T p.E1360K 0.453 NM_018557 c.G4078A Pair 31/32 1112 2 Positive Primary MCC26 chrX:53222732 KDM5C G A p.L1402F 0.126 NM_004187 c.C4204T NA nonpaired COSM169 4 Negative Primary MCC32 chr2:141641477 LRP1B C T p.E1360K 0.426 NM_018557 c.G4078A Pair 31/32 1112 4 Negative Metastasis MCC31 chr2:141643851 LRP1B C T p.D1274N 0.48 NM_018557 c.G3820A NA Pair 31/32 4 Negative Primary MCC32 chr2:141643851 LRP1B C T p.D1274N 0.415 NM_018557 c.G3820A NA Pair 31/32 1 Positive Primary MCC24 chr20:57429171 GNAS C T p.P284L 0.107 NM_080425 c.C851T NA nonpaired 2 Positive Primary MCC26 chr11:64572101 MEN1 C T p.G518E 0.137 NM_000244 c.G1553A NA nonpaired 4 Negative Primary MCC30 chr2:141773420 LRP1B C T p.A679T 0.494 NM_018557 c.G2035A NA nonpaired 2 Positive Primary MCC26 chr16:55519285 MMP2 G A p.G202R 0.111 NM_004530 c.G604A NA nonpaired 1 Positive Primary MCC24 chr16:15811182 MYH11 C T p.L1773L 0.107 NM_002474 c.G5319A NA nonpaired 4 Negative Metastasis MCC31 chr2:141812703 LRP1B C T p.D512N 0.464 NM_018557 c.G1534A NA Pair 31/32 4 Negative Primary MCC32 chr2:141812703 LRP1B C T p.D512N 0.448 NM_018557 c.G1534A NA Pair 31/32 4 Negative Primary MCC32 chr2:148672805 ACVR2A C T p.L192L 0.425 NM_001616 c.C574T NA Pair 31/32 4 Negative Metastasis MCC31 chr2:148672805 ACVR2A C T p.L192L 0.493 NM_001616 c.C574T NA Pair 31/32 4 Negative Primary MCC30 chr2:178098035 NFE2L2 G A p.Y115Y 0.386 NM_006164 c.C345T NA nonpaired

Downloaded From: https://jamanetwork.com/ on 09/27/2021 2 Positive Primary MCC26 chr22:28196321 MN1 G A p.H71Y 0.12 NM_002430 c.C211T NA nonpaired 4 Negative Primary MCC32 chr2:190660545 PMS1 C T p.I61I 0.463 NM_000534 c.C183T NA Pair 31/32 4 Negative Metastasis MCC31 chr2:190660545 PMS1 C T p.I61I 0.493 NM_000534 c.C183T NA Pair 31/32 4 Negative Primary MCC30 chr2:234681198 UGT1A1 C T p.T532I 0.502 NM_000463 c.C1595T NA nonpaired 4 Negative Primary MCC30 chr2:24933929 NCOA1 C T p.L850L 0.445 NM_003743 c.C2548T NA nonpaired 4 Negative Primary MCC30 chr2:60688300 BCL11A C T p.D583N 0.471 NM_018014 c.G1747A NA nonpaired 4 Negative Primary MCC30 chr2:60689366 BCL11A G A p.L227L 0.505 NM_018014 c.C681T NA nonpaired 4 Negative Primary MCC30 chr20:30253832 BCL2L1 C T p.E145K 0.45 NM_001191 c.G433A NA nonpaired 4 Negative Primary MCC30 chr20:31022784 ASXL1 C T p.Q757X 0.443 NM_015338 c.C2269T NA nonpaired 4 Negative Primary MCC30 chr22:28193278 MN1 G A p.P1085L 0.392 NM_002430 c.C3254T NA nonpaired 4 Negative Primary MCC32 chr22:36678800 MYH9 G C p.R1933G 0.393 NM_002473 c.C5797G NA Pair 31/32 4 Negative Metastasis MCC31 chr22:36678800 MYH9 G C p.R1933G 0.405 NM_002473 c.C5797G NA Pair 31/32 1 Positive Primary MCC24 chr5:180048576 FLT4 G A p.D662D 0.106 NM_002020 c.C1986 NA nonpaired 4 Negative Primary MCC30 chr22:41548001 EP300 GG AA p.E995K 0.436 NM_001429 c.2982_2983AA NA nonpaired p.T1909I/ 4 Negative Metastasis MCC31 chr22:41573441 EP300 CCC TTT 0.465 NM_001429 c.5726_5728TTT NA Pair 31/32 P1910S p.T1909I/ 4 Negative Primary MCC32 chr22:41573441 EP300 CCC TTT 0.406 NM_001429 c.5726_5728TTT NA Pair 31/32 P1910S 2 Positive Primary MCC26 chr9:139393449 NOTCH1 C T NA 0.118 NM_017617 c.6083-1G>A NA nonpaired 4 Negative Primary MCC30 chr3:47084125 SETD2 GG AA p.T2388I 0.416 NM_014159 c.7163_7164TT NA nonpaired 4 Negative Metastasis MCC31 chr4:103531776 NFKB1 G T p.D758Y 0.564 NM_003998 c.G2272T NA Pair 31/32 4 Negative Primary MCC32 chr4:103531776 NFKB1 G T p.D758Y 0.475 NM_003998 c.G2272T NA Pair 31/32 COSM167 198, COSM167 199, COSM167 4 Negative Primary MCC30 chr4:153244185 FBXW7 G A p.R578X 0.802 NM_018315 c.C1732T nonpaired 197, COSM142 7626, COSM229 67 4 Negative Primary MCC30 chr4:55141007 PDGFRA G A NA 0.46 NM_006206 c.1654-1G>A NA nonpaired 4 Negative Primary MCC30 chr4:55561706 KIT G A p.G32G 0.409 NM_000222 c.G96A NA nonpaired 1 Positive Primary MCC24 chr1:164529146 PBX1 G A p.G29G 0.106 NM_002585 c.G87A NA nonpaired 1 Positive Primary MCC24 chr2:212251691 ERBB4 C T p.S1123N 0.106 NM_005235 c.G3368A NA nonpaired 4 Negative Metastasis MCC31 chr5:112111421 APC C T p.P173L 0.349 NM_000038 c.C518T NA nonpaired 4 Negative Primary MCC30 chr5:138118976 CTNNA1 C T p.F72F 0.466 NM_001903 c.C216T NA nonpaired 4 Negative Metastasis MCC31 chr5:149433681 CSF1R C T p.G957E 0.449 NM_005211 c.G2870A NA Pair 31/32 4 Negative Primary MCC32 chr5:149433681 CSF1R C T p.G957E 0.485 NM_005211 c.G2870A NA Pair 31/32 4 Negative Metastasis MCC31 chr5:149441314 CSF1R C T p.W575X 0.535 NM_005211 c.G1725A NA Pair 31/32 4 Negative Primary MCC32 chr5:149441314 CSF1R C T p.W575X 0.397 NM_005211 c.G1725A NA Pair 31/32 4 Negative Metastasis MCC31 chr5:149459786 CSF1R C T p.V141M 0.483 NM_005211 c.G421A NA Pair 31/32 4 Negative Primary MCC32 chr5:149459786 CSF1R C T p.V141M 0.459 NM_005211 c.G421A NA Pair 31/32

Downloaded From: https://jamanetwork.com/ on 09/27/2021 2 Positive Primary MCC26 chr6:32185772 NOTCH4 C T p.G542R 0.157 NM_004557 c.G1624A NA nonpaired 2 Positive Primary MCC26 chr2:113999666 PAX8 C T p.D174N 0.105 NM_003466 c.G520A NA nonpaired 1 Positive Primary MCC23 chr6:135522857 MYB C T p.F556F 0.106 NM_005375 c.C1668T NA nonpaired 4 Negative Primary MCC30 chr5:180049747 FLT4 G A p.I547I 0.463 NM_002020 c.C1641T NA nonpaired 1 Positive Primary MCC24 chr9:133759974 ABL1 G A p.R766K 0.105 NM_005157 c.G2297A NA nonpaired 4 Negative Primary MCC30 chr5:180058740 FLT4 T C p.N33D 0.422 NM_002020 c.A97G NA nonpaired 4 Negative Metastasis MCC31 chr5:55237553 IL6ST G A p.P705L 0.452 NM_002184 c.C2114T NA Pair 31/32 4 Negative Primary MCC32 chr5:55237553 IL6ST G A p.P705L 0.422 NM_002184 c.C2114T NA Pair 31/32 2 Positive Primary MCC26 chr1:9780840 PIK3CD G A p.G521E 0.156 NM_005026 c.G1562A NA nonpaired 4 Negative Primary MCC32 chr6:117730754 ROS1 C T p.E94K 0.45 NM_002944 c.G280A NA Pair 31/32 3 Positive Primary MCC29 chr17:75484935 Sept9 C A p.A399A 0.271 NM_006640 c.C1197A NA nonpaired 4 Negative Metastasis MCC31 chr6:117730754 ROS1 C T p.E94K 0.446 NM_002944 c.G280A NA Pair 31/32 4 Negative Primary MCC32 chr6:152470714 SYNE1 C T p.L8109L 0.43 NM_033071 c.G24327A NA Pair 31/32 4 Negative Metastasis MCC31 chr6:152470714 SYNE1 C T p.L8109L 0.363 NM_033071 c.G24327A NA Pair 31/32 4 Negative Primary MCC32 chr6:152647481 SYNE1 C T p.R5010R 0.448 NM_033071 c.G15030A NA Pair 31/32 4 Negative Metastasis MCC31 chr6:152647481 SYNE1 C T p.R5010R 0.437 NM_033071 c.G15030A NA Pair 31/32 4 Negative Primary MCC30 chr6:152770727 SYNE1 C T p.G1156R 0.485 NM_033071 c.G3466A NA nonpaired 2 Positive Primary MCC26 chr20:39794394 PLCG1 C T p.T576I 0.192 NM_002660 c.C1727T NA nonpaired 2 Positive Primary MCC26 chr20:39794460 PLCG1 C T p.S598F 0.115 NM_002660 c.C1793T NA nonpaired 4 Negative Primary MCC30 chr6:32163710 NOTCH4 G A p.P1839L 0.103 NM_004557 c.C5516T NA nonpaired 4 Negative Primary MCC30 chr6:394995 IRF4 G A p.G131R 0.439 NM_002460 c.G391A NA nonpaired 4 Negative Metastasis MCC31 chr6:51613401 PKHD1 C T p.E3005K 0.468 NM_138694 c.G9013A NA Pair 31/32 1 Positive Primary MCC24 chr19:45868144 ERCC2 G A p.A182A 0.105 NM_000400 c.C546T NA nonpaired 4 Negative Primary MCC32 chr6:51613401 PKHD1 C T p.E3005K 0.458 NM_138694 c.G9013A NA Pair 31/32 4 Negative Metastasis MCC31 chr6:52876952 ICK G A p.S409F 0.461 NM_014920 c.C1226T NA Pair 31/32 4 Negative Primary MCC32 chr6:52876952 ICK G A p.S409F 0.453 NM_014920 c.C1226T NA Pair 31/32 4 Negative Metastasis MCC31 chr6:52878690 ICK G A p.L308L 0.504 NM_014920 c.C922T NA nonpaired 1 Positive Primary MCC24 chr7:98579568 TRRAP C T p.H2912H 0.105 NM_003496 c.C8736T NA nonpaired 1 Positive Primary MCC24 chr5:180057654 FLT4 C T p.V101I 0.105 NM_002020 c.G301A NA nonpaired 4 Negative Primary MCC30 chr6:69758197 BAI3 A T p.K743I 0.481 NM_001704 c.A2228T NA nonpaired 4 Negative Metastasis MCC31 chr6:70092728 BAI3 C T p.V1427V 0.484 NM_001704 c.C4281T NA Pair 31/32 4 Negative Primary MCC32 chr6:70092728 BAI3 C T p.V1427V 0.41 NM_001704 c.C4281T NA Pair 31/32 4 Negative Primary MCC30 chr6:93956626 EPHA7 C T p.L870L 0.451 NM_004440 c.G2610A NA nonpaired 4 Negative Metastasis MCC31 chr6:94120582 EPHA7 G A p.Q157X 0.429 NM_004440 c.C469T NA Pair 31/32 4 Negative Primary MCC32 chr6:94120582 EPHA7 G A p.Q157X 0.443 NM_004440 c.C469T NA Pair 31/32 4 Negative Primary MCC30 chr7:106509197 PIK3CG G A p.R397R 0.48 NM_002649 c.G1191A NA nonpaired 4 Negative Primary MCC30 chr7:106545800 PIK3CG G A p.G1093S 0.523 NM_002649 c.G3277A NA nonpaired

Downloaded From: https://jamanetwork.com/ on 09/27/2021 Also present in 32 but that one did not pass 4 Negative Metastasis MCC31 chr7:124465315 POT1 T A p.I595L 0.51 NM_015450 c.A1783T NA filtering due to low coverage

1 Positive Primary MCC24 chr22:28195917 MN1 G A p.G205G 0.105 NM_002430 c.C615T NA nonpaired chr13:11043593 1 Positive Primary MCC24 IRS2 G A p.Y823Y 0.104 NM_003749 c.C2469T NA nonpaired 2 4 Negative Primary MCC30 chr7:126173851 GRM8 C T p.V529M 0.448 NM_000845 c.G1585A NA nonpaired 4 Negative Primary MCC30 chr7:126544654 GRM8 C T p.E271K 0.437 NM_000845 c.G811A NA nonpaired RPS6KA 1 Positive Primary MCC24 chr6:166836847 G A p.P547L 0.104 NM_021135 c.C1640T NA nonpaired 2 1 Positive Primary MCC24 chrX:63411595 AMER1 G A p.C524C 0.103 NM_152424 c.C1572T NA nonpaired 1 Positive Primary MCC24 chr3:195591055 TNK2 G A p.R1038R 0.103 NM_005781 c.C3114T NA nonpaired 4 Negative Primary MCC30 chr7:126882975 GRM8 T C p.N95S 0.444 NM_000845 c.A284G NA nonpaired COSM120 4 Negative Metastasis MCC31 chr7:142561955 EPHB6 C T p.R133C 0.47 NM_004445 c.C397T Pair 31/32 5527 COSM120 4 Negative Primary MCC32 chr7:142561955 EPHB6 C T p.R133C 0.486 NM_004445 c.C397T Pair 31/32 5527 4 Negative Primary MCC30 chr7:151860732 KMT2C C T p.Q3310Q 0.424 NM_170606 c.G9930A NA nonpaired 4 Negative Primary MCC32 chr7:151876987 KMT2C G A p.F2458F 0.145 NM_170606 c.C7374T NA nonpaired 4 Negative Metastasis MCC31 chr7:151879061 KMT2C G A p.P1962S 0.923 NM_170606 c.C5884T NA nonpaired 4 Negative Primary MCC30 chr7:151932982 KMT2C G A p.R897X 0.433 NM_170606 c.C2689T NA nonpaired 1 Positive Primary MCC24 chr2:198261030 SF3B1 G A p.L1097F 0.103 NM_012433 c.C3289T NA nonpaired 4 Negative Primary MCC30 chr7:151947956 KMT2C G A p.P573S 0.421 NM_170606 c.C1717T NA nonpaired COSM145 4217, 4 Negative Primary MCC30 chr8:113326157 CSMD3 CT C p.K2454fs 0.533 NM_052900 c.7362delA nonpaired COSM145 4216 1 Positive Primary MCC24 chr19:40741976 AKT2 G A p.D332D 0.103 NM_001626 c.C996T NA nonpaired 1 Positive Primary MCC23 chr2:219563884 STK36 C T p.A1206V 0.102 NM_015690 c.C3617T NA nonpaired 1 Positive Primary MCC24 chr22:23656766 BCR G A p.K1197K 0.101 NM_004327 c.G3591A NA nonpaired COSM748 662, 4 Negative Metastasis MCC31 chr8:113331091 CSMD3 C T p.M2341I 0.5 NM_052900 c.G7023A Pair 31/32 COSM115 0924 2 Positive Primary MCC26 chr2:5833317 SOX11 G A p.G155D 0.107 NM_003108 c.G464A NA nonpaired COSM748 662, 4 Negative Primary MCC32 chr8:113331091 CSMD3 C T p.M2341I 0.523 NM_052900 c.G7023A Pair 31/32 COSM115 0924 4 Negative Metastasis MCC31 chr8:113516034 CSMD3 G A p.H1586Y 0.447 NM_052900 c.C4756T NA Pair 31/32 4 Negative Primary MCC32 chr8:113516034 CSMD3 G A p.H1586Y 0.45 NM_052900 c.C4756T NA Pair 31/32 4 Negative Metastasis MCC31 chr8:37696539 GPR124 C T p.P775P 0.476 NM_032777 c.C2325T NA Pair 31/32 4 Negative Primary MCC32 chr8:37696539 GPR124 C T p.P775P 0.515 NM_032777 c.C2325T NA Pair 31/32

Downloaded From: https://jamanetwork.com/ on 09/27/2021 4 Negative Metastasis MCC31 chr8:48706945 PRKDC G A p.P3526S 0.479 NA NA NA Pair 31/32 4 Negative Primary MCC32 chr8:48706945 PRKDC G A p.P3526S 0.492 NA NA NA Pair 31/32 3 Positive Primary MCC29 chr7:106508109 PIK3CG A C p.M35L 0.196 NM_002649 c.A103C NA nonpaired 4 Negative Primary MCC30 chr8:48734171 PRKDC A G p.F3035F 0.453 NA NA NA nonpaired 4 Negative Metastasis MCC31 chr9:134004760 NUP214 C T p.S163F 0.463 NM_005085 c.C488T NA Pair 31/32 4 Negative Primary MCC32 chr9:134004760 NUP214 C T p.S163F 0.455 NM_005085 c.C488T NA Pair 31/32 COSM110 4 Negative Metastasis MCC31 chr9:136913227 BRD3 G A p.P355L 0.502 NM_007371 c.C1064T Pair 31/32 6549 1 Positive Primary MCC24 chr5:180057025 FLT4 C T p.L198L 0.1 NM_002020 c.G594A NA nonpaired COSM110 4 Negative Primary MCC32 chr9:136913227 BRD3 G A p.P355L 0.444 NM_007371 c.C1064T Pair 31/32 6549 4 Negative Primary MCC30 chr9:139395215 NOTCH1 G A p.A1908V 0.467 NM_017617 c.C5723T NA nonpaired TNFRSF 2 Positive Primary MCC26 chr1:2489247 C T p.S51F 0.12 NM_003820 c.C152T NA nonpaired 14 4 Negative Metastasis MCC31 chr9:139400174 NOTCH1 G A p.L1392L 0.436 NM_017617 c.C4174T NA Pair 31/32 4 Negative Primary MCC32 chr9:139400174 NOTCH1 G A p.L1392L 0.344 NM_017617 c.C4174T NA Pair 31/32 4 Negative Primary MCC30 chr9:139401093 NOTCH1 T A NA 0.417 NM_017617 c.3902-2A>T NA nonpaired 4 Negative Primary MCC30 chr9:139418167 NOTCH1 A C NA 0.46 NM_017617 c.403+2T>G NA nonpaired 4 Negative Metastasis MCC31 chr9:32631678 TAF1L G A p.L1300L 0.477 NM_153809 c.C3900T NA Pair 31/32 4 Negative Primary MCC32 chr9:32631678 TAF1L G A p.L1300L 0.461 NM_153809 c.C3900T NA Pair 31/32 4 Negative Metastasis MCC31 chr9:35075303 FANCG GG AA p.R485W 0.522 NM_004629 c.1452_1453TT NA Pair 31/32 4 Negative Primary MCC32 chr9:35075303 FANCG GG AA p.R485W 0.496 NM_004629 c.1452_1453TT NA Pair 31/32 4 Negative Metastasis MCC31 chr9:37015068 PAX5 C T p.W112X 0.49 NM_016734 c.G336A NA Pair 31/32 4 Negative Primary MCC32 chr9:37015068 PAX5 C T p.W112X 0.44 NM_016734 c.G336A NA Pair 31/32 3 Positive Primary MCC29 chr14:92460166 TRIP11 A C p.V1716G 0.571 NM_004239 c.T5147G NA nonpaired 4 Negative Metastasis MCC31 chr9:5126756 JAK2 C T p.R1122X 0.442 NM_004972 c.C3364T NA Pair 31/32 4 Negative Primary MCC32 chr9:5126756 JAK2 C T p.R1122X 0.473 NM_004972 c.C3364T NA Pair 31/32 2 Positive Primary MCC26 chr16:2134482 TSC2 C T p.S1420L 0.118 NM_000548 c.C4259T NA nonpaired 4 Negative Primary MCC30 chr9:8465639 PTPRD G A p.R1181C 0.462 NM_002839 c.C3541T NA nonpaired 1 Positive Primary MCC24 chr8:37691637 GPR124 C T p.H533H 0.1 NM_032777 c.C1599T NA nonpaired 1 Positive Primary MCC24 chr6:31132577 POU5F1 C T p.R295Q 0.1 NM_002701 c.G884A NA nonpaired NM_000136:exo 4 Negative Metastasis MCC31 chr9:97873920 FANCC C T NA 0.5 c.1155-1G>A NA Pair 31/32 n14 4 Negative Primary MCC32 chr9:97873920 FANCC C T NA 0.477 NM_000136 c.1155-1G>A NA Pair 31/32 COSM961 59, COSM961 4 Negative Primary MCC30 chr9:98220325 PTCH1 G A p.F1046F 0.452 NM_000264 c.C3138T nonpaired 58, COSM961 60 4 Negative Metastasis MCC31 chrX:123505224 SH2D1A T G p.C124G 0.972 NM_002351 c.T370G NA Pair 31/32 4 Negative Primary MCC32 chrX:123505224 SH2D1A T G p.C124G 0.939 NM_002351 c.T370G NA Pair 31/32 4 Negative Primary MCC30 chrX:153762630 G6PD G T p.S219S 0.907 NM_000402 c.C657A NA nonpaired

Downloaded From: https://jamanetwork.com/ on 09/27/2021 All high confidence somatic non-synonymous and synonymous variants (see Methods) identified across 9 formalin-fixed, paraffin-embedded Merkel Cell Carcinoma samples are shown. The location (hg19), reference (REF) and variant (ALT) alleles, amino acid (AA) change, variant allele frequency (Var Allele Freq) are shown for each sample. AA: amino acid. MCV: Merkel cell polyomavirus.

Downloaded From: https://jamanetwork.com/ on 09/27/2021 eTable 4. Copy Number Alterations in Merkel Cell Carcinoma Cases

Sample Chromosome Change Category Note MCC24 1p gain nonpaired MCC25 1p loss Pair 25/26 MCC26 1p loss Pair 25/26 MCC25 1q gain Pair 25/26 MCC26 1q gain Pair 25/26 MCC28 1q gain nonpaired focal gain MARK1 MCC31 2p loss Pair 31/32 MCC32 2p loss Pair 31/32 MCC26 2q loss nonpaired MCC25 4p gain Pair 25/26 focal gain PHOX2B MCC26 4p gain Pair 25/26 focal gain PHOX2B MCC28 4q gain Pair 28/29 MCC29 4q gain Pair 28/29 MCC25 10 loss Pair 25/26 MCC26 10 loss Pair 25/26 MCC31 10p loss Pair 31/32 MCC32 10p loss Pair 31/32 MCC24 11 gain nonpaired MCC31 11p loss Pair 31/32 MCC32 11p loss Pair 31/32 MCC30 17p gain nonpaired MCC31 17q loss Pair 31/32 MCC32 17q loss Pair 31/32 MCC23 20q gain nonpaired MCC28 X gain Pair 28/29 MCC29 X gain Pair 28/29 MCC23 Xp loss nonpaired MCC24 Xq loss nonpaired

Downloaded From: https://jamanetwork.com/ on 09/27/2021 eTable 5. Primary-metastasis pairs used for similarity index comparison

CNA Mutation Clinical designation and sample similarity similarity Case number Site CNA Mutations index index Reference Primary (MCC32) Right cheek 4 64 Current 4 Right neck 1 0.91 Nodal metastasis (MCC31) 4 68 study LN

Primary (MCC14) Head 19 51 Harms et 5 0.41 0.09 Nodal metastasis (MCC9) LN 30 390 al 2016

Lower Primary (MCC16) 33 20 6 extremity Harms et 0.21 0.3 Lower al 2016 In-transit metastasis (MCC10) 10 12 extremity

CNA: copy number alteration.

Downloaded From: https://jamanetwork.com/ on 09/27/2021 eTable 6. Tumor purity estimates based on hematoxylin and eosin stained sections.

Merkel cell polyomavirus Observed mean allele Estimated % tumor Case# status frequency of all somatic purity mutations

MCC23 Positive 85% 20.0%

MCC24 Positive 95% 13.1%

MCC25 Positive 85% N/A*

MCC26 Positive 80% 13.5%

MCC28 Positive 70% 28.0%

MCC29 Positive 75% 34.0%

MCC30 Negative 85% 48.6%

MCC31 Negative 85% 51.4%

MCC32 Negative 80% 47.3%

*No somatic mutations were observed in MCC25.

Downloaded From: https://jamanetwork.com/ on 09/27/2021 Supplemental References

1. Harms PW, Collie AM, Hovelson DH, et al. Next generation sequencing of Cytokeratin 20-negative Merkel cell carcinoma reveals ultraviolet-signature mutations and recurrent TP53 and RB1 inactivation. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc. Mar 2016;29(3):240-248.

2. Kadakia KC, Tomlins SA, Sanghvi SK, et al. Comprehensive serial molecular profiling of an "N of 1" exceptional non-responder with metastatic prostate cancer progressing to small cell carcinoma on treatment. Journal of hematology & oncology. 2015;8(1):109.

3. Grasso C, Butler T, Rhodes K, et al. Assessing copy number alterations in targeted, amplicon-based next-generation sequencing data. The Journal of molecular diagnostics : JMD. Jan 2015;17(1):53-63.

4. Nemes S, Danielsson A, Parris TZ, et al. A diagnostic algorithm to identify paired tumors with clonal origin. Genes, chromosomes & cancer. Nov 2013;52(11):1007-1016.

5. Fisher CA, Harms PW, McHugh JB, et al. Small cell carcinoma in the parotid harboring Merkel cell polyomavirus. Oral surgery, oral medicine, oral pathology and oral radiology. Dec 2014;118(6):703-712.

6. Rodig SJ, Cheng J, Wardzala J, et al. Improved detection suggests all Merkel cell carcinomas harbor Merkel polyomavirus. The Journal of clinical investigation. Dec 2012;122(12):4645-4653.

7. Laude HC, Jonchere B, Maubec E, et al. Distinct merkel cell polyomavirus molecular features in tumour and non tumour specimens from patients with merkel cell carcinoma. PLoS pathogens. 2010;6(8):e1001076.

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