Cohen et al. 2014
Clinical Cancer Research
Supplement
Supplementary Methods:
Spectrum and relatedness of Human Papillomavirus in BRAFi-cSCC
The analysis was performed on the Phylogeny.fr platform (1) and comprised the following steps. HPV-L1 capsid protein sequences or partial sequences available from GenBank (Table S1 accession numbers analyzed) were extracted and aligned with MUSCLE (v3.7) configured for highest accuracy (2). The phylogenetic tree was reconstructed using the maximum likelihood method implemented in the PhyML program (v3.0 aLRT) (3). A graphical representation of the phylogeny tree was generated with TreeDyn (v198.3) (4) and was exported via PDF to Illustrator (CS3, Adobe Systems, San Jose CA) for visualization and annotation.
Putative novel HPV viral sequences were translated into all 6 possible reading frames (Emboss-Transeq <http://www.ebi.ac.uk/Tools/st/emboss_transeq/>) (5) and aligned to the L1 protein sequence of the BLAST-identified closest relative using T-COFFEE (ver. 10.00.r1613 build 432) (6).
Figure S1 – b-genus HPV38, HPV17 and HPV111 are most frequently found in BRAFi-cSCC
Figure S1: Relatedness of identified HPV genotypes from BRAF-inhibition induced adverse cutaneous squamous cell carcinomas during advanced melanoma therapy. Phylogenetic representation on the Phylogeny.fr platform with MUSCLE sequence alignment and TreeDyn rendering. The leaf widths are proportional to the number of patient samples with the genotype of HPV noted (legend). Samples where genotyping was unable to discriminate between two closely related genotypes both are listed with a ½ width leaf (e.g. SE68 & KC49, HPVx4b & RTRx4, FA39, & FA42). Boot strap percentage score (red numbers). *Putative novel HPV genotypes are depicted using the closest relative sequence available in GenBank (see supplemental alignment). Reference HPV (5, 6, 11, 16, 18) and bovine papillomavirus (BPV, 1, 5, 6) genotypes with grey branches and genus denoted in parentheses.
Figure S2: Protein and nucleic acid sequence alignments of putative novel HPV genotypes.
Amino acid alignment with homology: 70/80 = 87.5%
CRT-221YFPTVSGSLVTSDAQLFNRPFWLKRAQGHNNGVLWNNQIFVTVADNTRNTNFSISVAS58
HPV-100-L1333YFPTVSGSLVTSDAQLFNRPFWLKRAQGHNNGILWSNQMFVTVADNTRNTNFTISVTS390
cons333********************************:**.**:*************:***:*390
CRT-2259ENN-QQNYDAKNIREFMRHVEEY80
HPV-100-L1391EDLSTAKYDAKNIREYMRHVEEY413
cons391*::********:*******
Amino acid alignment with homology: 24/29 = 82.7%
CRT-11 1 FVTVVDTTRNTNFTISVTSQDGPLTEYDL29
HPV-107-L1339FVTVADNTRNTNFTISVPSQNGPLTEYDA367
cons313****.*.**********.**:*******367
Amino acid alignment with homology: 97/103 = 94.7%
CTR-11MANDVYGDACFFYARREQCYARHFFVRGGKPGDDIPADQIDAGKLKNEFYIPAEGGQA 58
HPV-118-L1236MQNDVYGDACFFYARREQCYARRFFVRGGKPGDDIPAEQIDAGKLKNEFYIPAAGGQA273
cons235*********************:**************:*******************273
CTR-159 QSQLGNSMYFPTVSGSLVSSDAQLFNRPFWLQRAQGHNNGILWNN------103
HPV-118-L1293QGQLGNSMYFPTVSGSLVSSDAQLFNRPFWLQRAQGHNNGILWGN 337
cons293*.*****************************************.* 337
1) CRT-22 using Megablast
Human papillomavirus partial L1 gene for major capsid protein, isolate SIBX20
Sequence ID: emb|HG326200.1|Length: 366Number of Matches: 1
Range 1: 1 to 138 GenBank
Alignment statistics for match #1
Score Expect Identities Gaps Strand
228 bits(123) 2e-56 133/138(96%) 0/138(0%) Plus/Minus
Query 2 ATATTCTTCTACATGTCTCATAAATTCCCTGATATTCTTGGCATCATAATTTTGTTGATT 61
||| ||||| ||||| |||||||||||||||||||| |||||||||||||||||||||||
Sbjct 138 ATACTCTTCAACATGCCTCATAAATTCCCTGATATTTTTGGCATCATAATTTTGTTGATT 79
Query 62 ATTTTCACTAGCTACACTGATACTAAAATTTGTGTTTCGTGTATTATCTGCTACAGTAAC 121
||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||
Sbjct 78 ATTTTCACTAGCTACACTGATACTAAAATTTGTGTTTCGTGTATTATCTGCTACAGTCAC 19
Query 122 AAATATTTGGTTATTCCA 139
||||||||||||||||||
Sbjct 18 AAATATTTGGTTATTCCA 1
2) CTR using Megablast
Human papillomavirus type 118 clone K2, complete genome
Sequence ID: gb|GQ246951.1|Length: 7597Number of Matches: 1
Range 1: 6513 to 6810 GenBank
Alignment statistics for match #1
Score Expect Identities Gaps Strand
351 bits(190) 2e-93 262/298(88%) 0/298(0%) Plus/Plus
Query 6 AAATGATGTATATGGTGATGCTTGCTTCTTTTATGCTAGACGGGAACAATGTTATGCACG 65
||||||||| ||||| ||||| ||||| |||||||||||||| || || |||||||| |
Sbjct 6513 AAATGATGTGTATGGCGATGCATGCTTTTTTTATGCTAGACGTGAGCAGTGTTATGCCAG 6572
Query 66 ACATTTCTTTGTTCGTGGTGGTAAGCCTGGGGATGACATACCAGCTGACCAAATAGATGC 125
|| || ||||||||||||||||||||||| ||||| || || || || |||||||||||
Sbjct 6573 ACGCTTTTTTGTTCGTGGTGGTAAGCCTGGTGATGATATTCCTGCAGAGCAAATAGATGC 6632
Query 126 AGGTAAACTGAAGAATGAATTTTATATACCAGCTGAAGGAGGTCAAGCCCAAAGTCAACT 185
|||||||| || |||||||| ||||||||||||| || || || || ||| |||||||
Sbjct 6633 TGGTAAACTTAAAAATGAATTCTATATACCAGCTGCGGGTGGGCAGGCTCAAGGTCAACT 6692
Query 186 TGGTAATTCAATGTATTTCCCAACTGTAAGTGGATCATTAGTATCTAGTGATGCTCAATT 245
||| ||||| ||||||||||||||||| ||||||||||||||||| |||||||| |||||
Sbjct 6693 TGGAAATTCCATGTATTTCCCAACTGTGAGTGGATCATTAGTATCGAGTGATGCCCAATT 6752
Query 246 ATTCAATAGGCCCTTCTGGCTACAAAGAGCACAGGGTCATAATAATGGCATTTTATGG 303
||| ||||||||||||||||||||||||||||||||||||||||||||||| ||||||
Sbjct 6753 ATTTAATAGGCCCTTCTGGCTACAAAGAGCACAGGGTCATAATAATGGCATCTTATGG 6810
3)
CRT-11 discontinguous megablast
Human papillomavirus isolate uwBCA34 major capsid protein L1 (L1) gene, partial cds
Sequence ID: gb|AF193303.1|AF193303Length: 238Number of Matches: 1
Range 1: 20 to 102GenBank
Alignment statistics for match #1
Score Expect Identities Gaps Strand
96.9 bits(106) 3e-17 71/83(86%) 0/83(0%) Plus/Minus
Query 18 TCATATTCTGTAAGTGGACCATCTTGAGAAGTAACACTGATAGTAAAATTGGTGTTTCTT 77
||||||||||||||||||| || |||| ||||||||| ||||||||||||||||| |||
Sbjct 102 TCATATTCTGTAAGTGGACTATTTTGACTAGTAACACTAATAGTAAAATTGGTGTTCCTT 43
Query 78 GTAGTATCTACCACAGTAACAAA 100
||| | ||| | || || |||||
Sbjct 42 GTATTGTCTGCAACTGTGACAAA 20
Table S1
HPV L1 sequences were obtained from NCBI for alignment and relatedness analysis. When sequencing identified two genotypes of equal likelihood (due to homology) the sample number for each is listed as 0.5.
Table S2 Human Papillomavirus Taxonomy
Nearest HPV genotype (% homology) / Taxonomy of nearest HPV / Patient (Specimen)CRT-11 / 107 (85%) / b2 / P441
CRT-22 / 100 (83%) / b1 / P281
CTR / 118 (88%) / b1 / P036
Known HPV isolates:
RTRX4: HPV118 (79% homology), HPV118 is b1 papillomavirus
HPVX4b: HPV118 (78%), HPV118 is b1 papillomavirus
SE68: HPV92 (83%), HPV92 is b4 papillomavirus
KC49: HPV92 (81%), HPV92 is b4 papillomavirus
SIBX18: HPV124 (87%), HPV124 is b1 papillomavirus
SIBX20: HPV120(87%), HPV120 is b2 papillomavirus
VS42-1: HPV23( 82%), HPV23 is b2 papillomavirus
VS20-4: HPV124 (81%), HPV124 is b1 papillomavirus
FA14: HPV124 (86%), HPV124 is b1 papillomavirus
FA132:HPV118 (80%), HPV118 is b1 papillomavirus
FA135: HPV21 (93%), HPV21 is b1 papillomavirus
FA108: HPV107(86%), HPV107 is b2 papillomavirus
FA118: HPV23(83%), HPV23 is b2 papillomavirus
FA18: HPV14 (78%), HPV14 is b1 papillomavirus
FA15: HPV164 (70%), HPV164 is g8 papillomavirus
FA39: HPV 96 (90%), HPV96 is b5 papillomavirus
FA42: HPV 96 (86%), HPV96 is b5 papillomavirus
FA7: HPV150 (81%), HPV150 is b5 papillomavirus
FA162: HPV96 (82%), HPV96 is b5 papillomavirus
Table S3: Ion Torrent Ampliseq panel targets
ABL1 / NM_007313.2 / 220-503
AKT1 / NM_005163.2 / 17, 32-52
ALK / NM_004304 / 1152,1171-1174,1196,1245,1275-1278
BRAF / NM_004333 / 464-469,592-601
CTNNB1 / NM_001904 / 32-45
DDR2 / NM_006182 / 768
EGFR / NM_005228 / 709-719, ex 19 del, 768-790, ex 20 ins, 833, 858-861
ERBB2 / NM_004448 / 310, 755-769, 774-780
FGFR1 / NM_000604 / 664, 656
FGFR2 / NM_000141.2 / 659, 544-550, 290, 372-376, 252-253
FGFR3 / NM_000142 / 248-249, 370-393, 650-652, 697
FGFR4 / NM_022963 / 535-550, 576, 641, 689
GNA11 / NM_002067.1 / 183, 209
GNAQ / NM_002072.2 / 183, 209
GNAS / NM_000516.3 / 201
HRAS / NM_005343 / 61, 12-13
IDH1 / NM_005896.2 / 132
IDH2 / NM_002168.2 / 140, 172
CKIT / NM_000222 / 417-421, 541-546, 550-576, 563-591, 642, 816-828, 829-850
KRAS / NM_004985 / 12-13, 61, 146
MAP2K1 / NM_002755 / 56-67, 121-124
MET / NM_000245 / 375, 982-988, 1010, 1112, 1248-1263
MPL / NM_005373.1 / 515
MYC / NM_002467.2 / 57-58, 131-138
NPM1 / NM_002520.4 / exon 12
NRAS / NM_002524 / 12-13, 61
PDGFRA / NM_006206 / 560-566, 659, 841-849
PIK3CA / NM_006218.1 / 88, 539-549, 1020-1025, 1043-1049
PIK3R1 / NM_181523.1 / 348, 440-463, 564-576
PTCH1 / NM_000264.2 / 688
RET / NM_020975 / 634, 883, 918
RUNX1 / NM_001754.4 / 344-371, 323-348, 291-323, 198-204, 162-166, 140-149, 106-114, 56
SMO / NM_005631.3 / 473, 533-535, 640, 652
STK11 / NM_000455 / 36-37, 60-66, 170-171, 194-199, 281-282, 354
WT1 / NM_024426.2 / 298-314, 390-397
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