xinpei yi, yuxing liao, kai li, bo wen, bing zhang

CaAtlas: an immunopeptidome atlas of human cancerXinpei Yi, Yuxing Liao, Kai Li, Bo Wen, Bing Zhang

Department of Molecular and Human Genetics, Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, USA

Abstract

• Human cancer antigen atlas (caAtlas) is a comprehensive resource developedthrough extensive collection and analysis of publicly available immunopeptidomedatasets from human cancer cell lines and tumor tissues from 9 different cancer types.

• To allow identification of putative tumor antigens inlcuding antigens with modifica-tions, we used an open search tool.

• In total, we identified almost 370,000 antigens, which is 60% more of HLA classI peptides than those in SysteMHC Atlas and tripled the number of HLA class IIpeptides.

• To identify neoantigens from the cancer immunopeptidomes, we developed Neo-Query on the basis of PepQuery.

• The antigens we identified included a substantial number of • neoantigens • C/Tantigens • cancer-associated antigens.

• Peptides from these verified antigen genes by caAtlas are potential therapeutic targetsfor cancer immunotherapy.

• We created a web resource named caAtlas (http://www.zhang-lab.org/caatlas/) to make all these data easily available and accessible to the broad cancerresearch community.

Construction of a comprehensive immunopeptidome atlasof human cancer

104

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D008984

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PXD

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PXD009751

PXD009749

PXD009750

PXD009753

PXD010808

PXD000394

PXD012083

PXD007935

PXD005704PXD010808PXD004746

PXD007635

PXD000394

PXD

009738

PX

D009752

PX

D009754

PX

D009755

PX

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008572

PXD

008571

PXD001898

PXD006215

PXD000394

PXD009738

PXD004233

PXD002439

PXD001205

PXD011723

PXD008500

PXD012348

PXD007679

PXD005231

PXD009531

PXD008127

PXD004023MSV000080527

Breast Cancer

Colon Carcinoma

Glioblastoma

Leuke

mia

Lung Cancer

Lym

phom

a

Melanoma

Meningiom

a

nonC

ance

r

Ovarian C

ancer

0 50000 100000 150000

nonCancer

Colon Carcinoma

Breast Cancer

Lung Cancer

Ovarian Cancer

Leukemia

Glioblastoma

Lymphoma

Melanoma

Meningioma

2000000 1500000 1000000 500000 0

#PSM #Peptide

1,052,731

1,026,991

672,414

295,497

287,743

109,585

75,811

71,188

14,409

2,152,328

164,717

111,118

69,900

43,509

40,720

8,368

17,154

17,128

4,392

158,658

8,189,718 5,099,093

4,001,889

2,8

00,3

93

2,7

99,2

66

2,381,468

706,337

589,883152,872

18,9

91,0

18

45,711,937 MS/MS

42 datasets

32 Class I, 3 Class II, 7 both

45.7M MS/MS

Open Search

5.8M PSMs (FDR<1%)

364,283 peptides (FDR<1%)

on 14,858 proteins to 16,059 proteins

#P

ep

tid

e

Length (a.a)

Dataset publication time

Cu

mu

lative

#p

ep

tid

es

A B

C

F G

102,516 26,488 13,554

caAtlas

SysteMHC Atlas

SysteMHC Atlas

caAtlas

ED

143,986 112,587 46,478

Class I Class II

Figure 1:(A). MS/MS spectra numbers of the immunopeptidomics datasets included in this study andsummary peptide identification results.(B). The distributions of PSM and peptide corresponding to 9different cancer types and non-cancerous samples, respectively. (C). Typical length distribution of HLAclass I and HLA class II peptides. (D) Comparison of the HLA class I peptide numbers between this studyand SysteMHC Atlas. (E) Comparison of the HLA class II unique peptide numbers between this studyand SysteMHC Atlas. (F) Cumulative number of all distinct HLA class I peptides as a function of datasetpublication time. Each dataset is denoted as dataset ID: dataset public time (#peptides).(G) Cumulativenumber of distinct peptides for each of the top nine HLA class I alleles as a function of dataset publicationtime.

Neoantigens & C/T antigens & Cancer-associated antigens verified by caAtlas

Neoantigens

AADAC_S285F (HLA-C*07:02, 0.01%) • AQP7_A8T (HLA-A*03:01,HLA-A*68:01, 0.04%)ATAD3B_F86L (HLA-B*15:17,HLA-C*15:05, 0.03%)ATP2B2_R592C (HLA-A*03:01, 0.03%)C11orf68_Q154R (HLA-A*11:01, 0.03%)CASC5_R43T (HLA-A*11:01,HLA-A*68:01, 0.02%) • CD86_A97T (HLA-A*03:01, 0.02%)CMYA5_A3318V (HLA-B*08:01, 0.02%)COG4_T85I (HLA-B*44:02, 0.03%)COL14A1_R226Q (HLA-A*02:05,HLA-C*12:03, 0.02) • CRB1_L670F (HLA-A*68:01, 0.01%) • DCAF4L1_R125Q (HLA-B*07:02, 0.02%)DGKI_R723C (HLA-B*08:01, 0.04%) • F7_T350M (HLA-A*02:01,HLA-A*02:05,HLA-A*02:06, 0.02%)FANK1_A147T (HLA-B*07:02, 0.02%)FCGBP_D4906H (HLA-A*02:01, 0.04%)GADL1_G206V (HLA-B*08:01, 0.02%)GALP_V6I (HLA-B*07:02, 0.02%)GFPT2_T15M (HLA-B*07:02,HLA-B*08:01, 0.03%)GRM5_S71Y (HLA-A*24:02, 0.01%)HADH_L145P (HLA-A*11:01,HLA-B*44:02, 0.03%)HCRTR2_K370I (HLA-A*24:02, 0.01%)HGFAC_D595N (HLA-A*03:01, 0.01%)IGHV1OR21−1_T110K (HLA-A*01:01, 0.02%)KCNJ3_R439Q (HLA-B*51:01, 0.02%)LCP1_K102E (HLA-B*49:01, 0.05%)MOBP_E20K (HLA-A*03:01,HLA-A*11:01, 0.01%)NAGA_R287L (HLA-A*02:01, 0.01%)PCDHA3_E253K (HLA-A*03:01, 0.03%) • PCDHGB7_A509V (HLA-A*31:01,HLA-A*68:01, 0.01%)PLAG1_S197L (HLA-A*03:01, 0.01%)POM121L12_A270V (HLA-A*02:01,HLA-A*68:01, 0.04%) • • PPFIBP2_R500G (HLA-A*03:01, 0.03%)PXDNL_G382E (HLA-B*15:01,HLA-C*12:03, 0.01%)RIMS1_S582L (HLA-B*07:02, 0.01%)SACS_L328F (HLA-A*24:02,HLA-A*68:01, 0.01%)SELP_R56C (HLA-A*01:01, 0.01%)SORCS3_S95L (HLA-A*02:01,HLA-A*02:06,HLA-B*07:02, 0.03%) • SPCS1_P41A (HLA-A*68:01, 0.05%)STOM_D17N (HLA-A*68:01, 0.01%)SYNE2_D3286H (HLA-B*35:01, 0.04%) • SZT2_P1826L (HLA-A*02:01,HLA-A*68:01, 0.01%)TCEB1_T41M (HLA-A*02:01,HLA-C*12:03, 0.02%)TNNC1_D44N (HLA-A*03:01, 0.02%) • TPTE_P448S (HLA-A*01:01, 0.02%)TPTE_T273N (HLA-B*07:02, 0.01%)TROAP_G280D (HLA-A*68:01, 0.01%)UBR4_R849C (HLA-A*02:01,HLA-A*02:20, 0.01%)USE1_L113S (HLA-A*68:01, 0.03%)ZNF600_S161F (HLA-A*23:01,HLA-A*24:02, 0.02%)C5AR2_V122I (HLA-A*02:01,HLA-A*02:20, 0.01%)CHRD_T339M (HLA-A*03:01, 0.01%)COL6A3_T2868I (HLA-A*03:01, 0.06%)ECEL1_R296Q (HLA-C*07:02,HLA-C*12:03, 0.02%)ELP2_Q153H (HLA-A*24:02,HLA-A*32:01, 0.01%)GPR112_D217Y (HLA-B*15:01,HLA-B*58:01, 0.01%)GRIN2D_S644L (HLA-A*68:01, 0.01%)GRM8_D145N (HLA-B*07:02, 0.02%)IL37_E122K (HLA-A*03:01, 0.02%)LILRB4_Q414R (HLA-A*03:01,HLA-A*68:01, 0.02%)MYH3_D718N (HLA-A*03:01, 0.01%) • PLEC_S2677P (HLA-B*07:02, 0.03%)RRBP1_R665L (HLA-A*02:01, 0.04%)SLC35A2_A81V (HLA-A*02:01, HLA-C*03:04, 0.01%)TREX2_V56I (HLA-A*02:01, 0.01%)ZFP42_T270M (HLA-A*03:01,HLA-A*68:01, 0.02%)CDKN2A_Q50R (HLA-A*31:01,HLA-B*07:02,HLA-C*07:02, 0.01%)CUEDC1_E321K (HLA-A*03:01, 0.01%)DSCAML1_V1182I (HLA-A*03:01, 0.02%) • • ENTPD6_K94E (HLA-A*02:01,HLA-A*03:01,HLA-B*40:01, 0.03%)ITGB2_Q354H (HLA-A*68:01,HLA-B*15:01,HLA-B*40:01, 0.03%)NAALADL2_S393L (HLA-B*07:02, 0.01%) • PRDM9_D871N (HLA-A*68:01, 0.02%)PVR_E6K (HLA-A*03:01, 0.02%)HMGXB3_A456V (HLA-B*07:02; 0.02%)

0 2 4 6 8#Sample

CancerTypeBreastCancer

ColonCarcinoma

Glioblastoma

Leukemia

LungCancer

Lymphoma

Melanoma

Meningioma

OvarianCancer

HMGXB3_A456V (HLA-B*07:02,9.68nM)

ITGB2_Q354H (HLA-A*68:01,24.65nM)

ANKLE2_G246A (HLA-B*35:08,36.68nM)

A B

Figure 2:Neoantigens verified by HLA class I immunopep-tidome of cancer samples.(A) Somatic mutations supportedby immunopeptidomics data from three or more tumorsamples. The identified HLA Alleles and the frequenciesof these mutations in ICGC are list on the left. (B) Anno-tated spectra of the three neoantigen peptides in (A).

C/T antigens

DAZL • •

DDX53 •

FATE1 •

HORMAD1 •

MAEL •

MAGEB1 •

PAGE1 • •

PASD1 • •

PIWIL2 •

SPAG6 •

SPANXB1 •

SPANXN2 •

SPERT • •

SSX1 • •

SSX4 • •

SYCP1 • •

CCDC62 •

CSAG1 •

CT47A1 •

CTAG1A • •

IGSF11 •

LY6K •

MAGEA10 •

PAGE2 •

PAGE5 •

SSX2 • •

SSX3 • •

SYCE1 •

TDRD1 •

ACRBP •

IL13RA2 •

RGS22 •

GAGE5 •

SPEF2 •

MAGEC1 • •

MROH2B • •

SPAG4 •

MAGEA11 •

MAGEC2 • •

SPAG17 •

ANKRD45 •

ROPN1 •

0 5 10

#Sample

CancerTypeBreastCancer

Glioblastoma

Leukemia

LungCancer

Lymphoma

Melanoma

Meningioma

OvarianCancer

CT

antig

ens d

atabase

Testis sp

ecific pro

teins

Adipose - Subcutaneous

Adipose - Visceral (Omentum)

Adrenal Gland

Artery - Aorta

Artery - Coronary

Artery - Tibial

Bladder

Brain - Amygdala

Brain - Anterior cingulate cortex (BA24)

Brain - Caudate (basal ganglia)

Brain - Cerebellar Hemisphere

Brain - Cerebellum

Brain - Cortex

Brain - Frontal Cortex (BA9)

Brain - Hippocampus

Brain - Hypothalamus

Brain - Nucleus accumbens (basal ganglia)

Brain - Putamen (basal ganglia)

Brain - Spinal cord (cervical c-1)

Brain - Substantia nigra

Breast - Mammary Tissue

Cells - Cultured fibroblasts

Cells - EBV-transformed lymphocytes

Cervix - Ectocervix

Cervix - Endocervix

Colon - Sigmoid

Colon - Transverse

Esophagus - Gastroesophageal Junction

Esophagus - Mucosa

Esophagus - Muscularis

Fallopian Tube

Heart - Atrial Appendage

Heart - Left Ventricle

Kidney - Cortex

Kidney - Medulla

LiverLung

Minor Salivary Gland

Muscle - Skeletal

Nerve - Tibial

OvaryPancreas

Pituitary

Prostate

Skin - Not Sun Exposed (Suprapubic)

Skin - Sun Exposed (Lower leg)

Small Intestine - Terminal Ileum

SpleenStomach

TestisThyroid

UterusVagina

Whole Blood

0

20

40

60

80

100

120

TP

M

Gene expression for MROH2B (ENSG00000171495.16)

Adipose - Subcutaneous

Adipose - Visceral (Omentum)

Adrenal Gland

Artery - Aorta

Artery - Coronary

Artery - Tibial

Bladder

Brain - Amygdala

Brain - Anterior cingulate cortex (BA24)

Brain - Caudate (basal ganglia)

Brain - Cerebellar Hemisphere

Brain - Cerebellum

Brain - Cortex

Brain - Frontal Cortex (BA9)

Brain - Hippocampus

Brain - Hypothalamus

Brain - Nucleus accumbens (basal ganglia)

Brain - Putamen (basal ganglia)

Brain - Spinal cord (cervical c-1)

Brain - Substantia nigra

Breast - Mammary Tissue

Cells - Cultured fibroblasts

Cells - EBV-transformed lymphocytes

Cervix - Ectocervix

Cervix - Endocervix

Colon - Sigmoid

Colon - Transverse

Esophagus - Gastroesophageal Junction

Esophagus - Mucosa

Esophagus - Muscularis

Fallopian Tube

Heart - Atrial Appendage

Heart - Left Ventricle

Kidney - Cortex

Kidney - Medulla

LiverLung

Minor Salivary Gland

Muscle - Skeletal

Nerve - Tibial

OvaryPancreas

Pituitary

Prostate

Skin - Not Sun Exposed (Suprapubic)

Skin - Sun Exposed (Lower leg)

Small Intestine - Terminal Ileum

SpleenStomach

TestisThyroid

UterusVagina

Whole Blood

0

100

200

300

400

TP

M

Gene expression for SPERT (ENSG00000174015.9)

Adipose - Subcutaneous

Adipose - Visceral (Omentum)

Adrenal Gland

Artery - Aorta

Artery - Coronary

Artery - Tibial

Bladder

Brain - Amygdala

Brain - Anterior cingulate cortex (BA24)

Brain - Caudate (basal ganglia)

Brain - Cerebellar Hemisphere

Brain - Cerebellum

Brain - Cortex

Brain - Frontal Cortex (BA9)

Brain - Hippocampus

Brain - Hypothalamus

Brain - Nucleus accumbens (basal ganglia)

Brain - Putamen (basal ganglia)

Brain - Spinal cord (cervical c-1)

Brain - Substantia nigra

Breast - Mammary Tissue

Cells - Cultured fibroblasts

Cells - EBV-transformed lymphocytes

Cervix - Ectocervix

Cervix - Endocervix

Colon - Sigmoid

Colon - Transverse

Esophagus - Gastroesophageal Junction

Esophagus - Mucosa

Esophagus - Muscularis

Fallopian Tube

Heart - Atrial Appendage

Heart - Left Ventricle

Kidney - Cortex

Kidney - Medulla

LiverLung

Minor Salivary Gland

Muscle - Skeletal

Nerve - Tibial

OvaryPancreas

Pituitary

Prostate

Skin - Not Sun Exposed (Suprapubic)

Skin - Sun Exposed (Lower leg)

Small Intestine - Terminal Ileum

SpleenStomach

TestisThyroid

UterusVagina

Whole Blood

0

50

100

150

TP

M

Gene expression for DAZL (ENSG00000092345.13)

A B

C

D

Figure 3:42 CT antigens from the CT antigens databaseand the GTEx-supported testis-specific proteins with theantigen source genes in caAtlas identified in tumor samplesbut not normal samples. The gene expression distributionamong all the 54 normal tissues from GTEx for the 3 pre-viously unrecognized CT antigens.

Cancer-associated antigens

Area (%)Gene count

ANO4ART3

BAALCCCL19

CD300LDCHRNA1

CSAG1 CTAG1A

FAM178BFBLN7FGF13

IGSF11 IL12RB2KCNK3LRFN4

MAGEA10 MDGA2

NANOS1NANPNBL1

NR0B1NTM

PAGE5 PDXP

PI15PTCHD4RAMP1RNF128SCUBE2SHISA2

SLC7A10SORCS1

SOSTDC1SULT1C2

THEM4TRIM48TRIQK

XAGE1ACCL18

DNASE2BFAM210BGPR158

IGLV3−12NOL4L

NUDT17RBP5SDSL

SLC24A4BFSP1

GORABKLHL38MMP15

SLITRK2CYP11B1MAGEC1

RLBP1SOX6

STK32ATRIM51TRIM63

UCN2TMEM144TSPAN10

TYRP1MAGEC2

MLANAST3GAL6

KCNJ5SLC45A2

DCTROPN1

ROPN1B TYR

0 5 10 15#Sample

0 10000 20000 30000 40000 50000Sig

n(L

og2F

C)

x log10(p

valu

e)

200

-200

0

100

Fre

quency o

f sam

ple

s (

%)

50

0

50

100

Melanoma

nonMelanoma

•

• •

• •

•

•

•

• • •

• •

• •

•

•

CT

an

tigen

s d

ata

base

Testis

sp

ecific

pro

tein

s

A

B

C

Figure 4:Differential analysis of antigens betweenmelanoma samples and non-melanoma samples. Genenames for the 73 melanoma-associated genes which aresignificantly higher expression in the TCGA melanomasamples compared to GTEx normal skin tissues andsorted by sample numbers.

Open-pFind showed relatively higher sensitivityand specificity

4719

23552235

17551596

13471201

966851827

633628613610562561556473414400335300286236215194187186173160155153117103 95 93 78 77 65 64

0

1000

2000

3000

4000

5000

Res

ults

of s

ix d

iffer

ent s

earc

h en

gine

s

open-pFind

SysteMHC Atlas

MS-GF+

Comet

MaxQuant

X!Tandem

05000100001500020000#Peptides

80%

76%

85%

90%

93%

2125

1091

873867866811

750

625578573563539520490478

350317

256248195195194186181159148138135132127124121

69 56 51 45 41 37 32 310

500

1000

1500

2000

Res

ults

of s

ix d

iffer

ent s

earc

h en

gine

s

open-pFind

SysteMHC Atlas

Comet

MaxQuant

MS-GF+

X!Tandem

0300060009000#Peptides

70%

76%

80%

70%

91%

A B

2020

518

897

1685

615

420

1251

0

7777

1064

5

8162

7964

6058

5464

1965

Overlap

Open−pFind

SysteMHC Atlas

MS−GF+

Comet

MaxQuant

X!Tandem

100 75 50 25 00 25 50 75 100

82(3857/4719)

79(15914/20205)

78(14806/18897)

81(13684/16856)

79(12215/15420)

80(9953/12510)

80(6218/7777)

31(29/93)

58(272/473)

54(161/300)

78(1373/1755)

57(772/1347)

71(1661/2355)

82(3857/4719)

HLA-binder percentage (%)

Total identifications Unique identifications

Overlap

Open−pFind

SysteMHC Atlas

MS−GF+

Comet

MaxQuant

X!Tandem

100 75 50 25 00 25 50 75 100

HLA-binder percentage (%)

Total identifications Unique identifications

84(1641/1965)

82(4983/6058)

83(6587/7964)

81(4449/5464)

84(6852/8162)

83(8845/10645)

84(735/873)

35(13/37)

68(391/573)

71(412/578)

76(655/867)

77(837/1091)

80(1693/2125)

84(735/873)

Mel 15 Mel 16

C D

Figure 5:Open-pFind produces relatively higher sensitivity and specificity compared tofour other search engines and SysteMHC Atlas in peptide identification from HLA classI data, and thus was selected for further analyses in this study.

Data dissemination through caAtlas

Figure 6:CaAtlas web resource: http://www.zhang-lab.org/caatlas/. All antigensare searchable by gene symbol, protein name, or protein ID. Moreover, neoantigens, CTantigens, and cancer-associated antigens, are browsable in three separate lists, in whichthe antigen source genes can be sorted by sample number or associated cancer typeinformation. Conclusion

• CaAtlas expanded immunopeptidomics validated neoantigens from a fewdozens in the existing literature to more than 3,000.

• CaAtlas provided direct evidence to support tumor specific-presentationof some known and putative novel CT antigens and also revealed non-tumor-specific presentation of some previously annotated CT antigens.

• CaAtlas provided direct mass spectrum evidence to support known andputative novel tumor-associated differentiation or overexpressed antigengenes.

• CaAtlas website allows users to manually check and download the massspectrum matching results for all antigens identified in this study.

Acknowledgements: This study was supported by the Cancer Prevention and Research Institute of Texas (CPRIT) award RR160027, and funding from the McNair Medical Institute at The Robert and Janice McNair Foundation.