Supplementary Information (Tables)
Protein signatures correspond to survival outcomes of AJCC stage III melanoma patients
Swetlana Mactier1*, Kimberley L. Kaufman1, Penghao Wang2, Ben Crossett1, Gulietta M. Pupo3,4, Philippa L. Kohnke1, John F. Thompson4,6,8, Richard A. Scolyer4,5,7, Jean Y. Yang2, Graham J. Mann3,4, Richard I. Christopherson1
Table S1. Clinico-pathologic characteristics of 33 AJCC stage III melanoma patients and their tumors
Variable / Poor survival group (n = 14) / Good survival group (n = 19) / Good vs poor (p-value)aAge (years, median) / 61.6 / 60.2 / 0.38
Sex (% Female) / 42.9% / 42.1% / 1.00
Survival (days, median) / 189.5 / 2939 / n/a
Survival (days, range) / 27-344 / 1499-4357 / n/a
Previous primary melanoma
Body site (% severe sun damage) / 28.6% / 42.1% / 0.43
Stage at diagnosis (% stage I or II) / 71.4% / 73.7% / 0.18
Breslow thickness (median, mm) / 2.6 / 2.9 / 0.74
Mitotic rate (median , mm2) / 6.3 / 5.1 / 0.66
Melanoma sub type (% with a nodular component) / 21.4% / 21.1% / 0.33
Presence of regression / 57.1% / 36.8% / 0.26
Ulceration / 30.8% / 6.7% / 0.13
Nodal tumor analyzed
Metastasis size (median, mm) / 37.5 / 35 / 0.45
Extranodal spread / 50% / 21.1% / 0.14
Number of invasive nodes (median) / 10 / 2 / 0.05
Cell size (2, large) / 57.1% / 68.4% / 0.21
Pigmentation (present) / 42.9% / 15.8% / 0.07
Mutational status
BRAF / 42.9% / 47.4% / 1.00
NRAS / 28.6% / 36.8% / 0.72
PIK3CA / 7.1% / 0% / 0.42
cKIT / 0% / 0% / n/a
FLT3 / 0% / 5.3% / 1.00
MET / 0% / 0% / n/a
PDGFRA / 7.1% / 0% / 0.42
EGFR / 0% / 0% / n/a
a p-value was calculated by Fisher’s exact test (R statistical environment), except for: age, number of invasive nodes (Student’s t-test, Excel), Breslow thickness, mitoses and metastasis size (Mann-Whitney, R statistical environment); n/a, not applicable.
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Table S2. Melanoma patient demographics, pathology, lymph node recurrence and survival parameters
Demographics / Culprit primary melanoma / Lymph node metastases / PrognosisNo. / Sex / Age / Site / T Stage / Breslow
thickness
in mm / Mitotic rate / mm2 / Clark level / Histology / Ulc. / AJCC
Stage / No. of Nodes / Size
in mm / Extranodal spread / Survival
(days)
Patients with good prognosis
1 / M / 61 / Lower Arm / T1 / 0.4 / 0 / 2 / SSM / No / Stage III / 10 / 120 / no / 4357
2 / F / 44 / Occult / T0 / NA / NA / NA / Not Known / NA / Stage III / 1 / 24 / yes / 3921
3 / F / 64 / Lower Leg / T4b / 4.5 / 21 / 4 / NM / No / Stage III / 2 / 20 / no / 3650
4 / M / 44 / Chest / T3a / 3 / 8 / 3 / SSM / No / Stage III / 1 / 38 / no / 3537
5 / F / 75 / Lower Leg / T2 / 1.5 / 0 / 4 / SSM / No / Stage III / 1 / 35 / no / 3528
6 / F / 57 / Upper Arm / T2 / 1.42 / 5 / 3 / SSM / No / Stage III / 1 / 25 / no / 3509
7 / F / 22 / Upper Leg / T4a / 5.2 / 8 / 4 / NM (Spitz naevus) / No / Stage III / 2 / 65 / no / 3357
8 / F / 40 / Lower Leg / T2 / 1 / 2 / 3 / SSM / No / Stage III / 1 / 35 / no / 2974
9 / M / 64 / Back / T1 / 0.7 / 0 / 2 / NM / No / Stage III / 4 / 45 / no / 2969
10 / M / 51 / Lower Leg / T4a / 4.2 / 5 / 4 / SSM / Yes / Stage III / 4 / 15 / no / 2939
11 / M / 40 / Occult / T0 / NA / NA / NA / Not Known / NA / Stage III / 5 / 25 / no / 2932
12 / M / 52 / Upper Leg / T2 / 0.85 / 10 / 4 / Not Known / No / Stage III / 1 / 35 / yes / 2893
13 / F / 67 / Lower Leg / T2 / 0.8 / 0 / 3 / SSM / No / Stage III / 7 / 40 / yes / 2764
14 / M / 30 / Upper Arm / T4a / 6.2 / 8 / 5 / SSM / No / Stage III / 2 / 30 / yes / 2408
15 / M / 30 / Occult / T0 / NA / NA / NA / Not Known / NA / Stage III / 1 / 95 / no / 2115
16 / M / 63 / Back / T4b / 11 / 8 / 5 / NM / No / Stage III / 3 / 25 / no / 1929
17 / M / 66 / Back / T1 / 0.55 / 0 / 2 / SSM / No / Stage III / 3 / 60 / no / 1913
18 / F / 63 / Upper Arm / T3a / 2.5 / 2 / 4 / SSM / No / Stage III / 1 / 50 / no / 1888
19 / M / 47 / Occult / T0 / NA / NA / NA / Not Known / NA / Stage III / 1 / 40 / no / 1499
Patients with poor prognosis
20 / F / 53 / Chest / T3a / 2.2 / 2 / 4 / SSM / No / Stage III / 9 / 58 / yes / 344
21 / F / 57 / Lower Leg / T3a / 1.7 / 1 / 3 / SSM / No / Stage III / 2 / 35 / no / 326
22 / M / 56 / Lower Leg / T3a / 3 / 13 / 3 / NM / Yes / Stage III / 20 / 25 / no / 275
23 / M / 75 / Back / T1 / 0.6 / 3 / 2 / SSM / No / Stage III / 20 / 50 / yes / 235
24 / M / 67 / Chest / T3b / 3.4 / 2 / 5 / NM / No / Stage III / 11 / 50 / yes / 215
25 / M / 77 / Acral - Toe / T4a / 6.75 / 25 / 5 / Acral Lentignious / Yes / Stage III / 1 / 40 / no / 193
26 / F / 74 / Upper Leg / T2 / 1.36 / 5 / 4 / SSM / No / Stage III / 6 / 70 / yes / 190
27 / M / 54 / Upper Leg / T4a / 4.5 / 1 / 4 / SSM / Yes / Stage III / 2 / 25 / no / 189
28 / F / 57 / Back / T3a / 1.8 / 4 / 4 / NM / No / Stage III / 63 / 15 / no / 183
29 / F / 46 / Occult / TX / NA / NA / NA / Not Known / NA / Stage III / 4 / 80 / yes / 176
Table S2. continued
Demographics / Culprit primary melanoma / Lymph node metastases / Survival
No. / Sex / Age / Site / T Stage / Breslow
thickness
in mm / Mitotic rate / mm2 / Clark level / Histology / Ulc. / AJCC
Stage / No. of Nodes / Size
in mm / Extranodal spread / Prognosis
(days)
30 / M / 69 / Chest / T3a / 2.2 / 2 / 3 / SSM / No / Stage III / 16 / 30 / yes / 70
31 / M / 53 / Acral - Sole / T3a / 2 / 1 / 4 / Acral Lentiginous / No / Stage III / 3 / 50 / no / 67
32 / F / 78 / Lower Leg / T3a / 2.7 / 20 / 4 / SSM / Yes / Stage III / 13 / 25 / yes / 43
33 / M / 65 / Lower Arm / T3a / 2 / 3 / 4 / NM / No / Stage III / 40 / 30 / no / 27
Additional patient cohort (used for SRM analysis)
34 / M / 75 / Hand / T2 / 1 / 2 / 3 / NM / No / Stage III / 10 / 60 / yes / 1445
35 / M / 28 / Back / T1 / 0.7 / 0 / 3 / SSM / Yes / Stage III / 1 / 35 / no / 1326
36 / M / 76 / Back / TX / 1.6 / NA / NA / NM / NA / Stage IV / 24 / 40 / yes / 1286
37 / M / 60 / Chest / T2 / 1 / 0 / 3 / SSM / No / Stage III / 1 / 60 / no / 752
38 / M / 59 / Acral - Toe / T3b / 4 / 6 / 5 / Acral Lentiginous / Yes / Stage III / 2 / 32 / yes / 714
39 / M / 66 / Chest / T2 / 1 / 0 / 3 / SSM / No / Stage III / 4 / 40 / yes / 454
40 / M / 59 / Lower Arm / T3a / 2.2 / 6 / 4 / SSM / No / Stage IV / 3 / 47 / no / 430
41 / M / 52 / Occult / T0 / NA / NA / NA / Not Known / NA / Stage IV / 5 / 60 / yes / 408
42 / M / 77 / Back / T3a / 2.3 / 9 / 3 / SSM / Yes / Stage IV / 7 / 25 / yes / 384
43 / M / 47 / Lower Leg / TX / NA / NA / NA / NA / NA / Stage IV / 1 / 85 / no / 380
Abbreviations: AJCC, American Joint Committee on Cancer; NA, not applicable, NM, nodular melanoma; SSM, superficial spreading melanoma; Ulc, ulceration. T0, no evidence of primary melanoma, TX, primary melanoma cannot be assessed.
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Table S3. Differentially abundant proteins between poor and good prognosis AJCC stage IIIc melanoma patients identified using DIGE
Spot no.a / Access. nob / Protein name / Gene name / Normalized abundancec / FDdPP/GP / p- valuee / Mascot
score / Sequence coverage % / No. of peptides matched
GP / PP
1 / P02452 / Collagen alpha-1(I) chain / COL1A1 / 0.60 / 1.60 / 2.67 / 0.001 / 218 / 2 / 4
3 / Q59FC6 / Tumour rejection antigen (GP96) 1 variant / HSP90B1 / 0.63 / 1.29 / 2.05 / 0.017 / 648 / 18 / 16
19 / Q59FC6 / Tumor rejection antigen (GP96) 1 variant / HSP90B1 / 0.80 / 1.18 / 1.48 / 0.042 / 726 / 19 / 14
5 / P09211 / Glutathione S-transferase P / GSTP1 / 0.73 / 1.23 / 1.69 / 0.011 / 246 / 21 / 3
13 / P09211 / Glutathione S-transferase P / GSTP1 / 0.91 / 1.18 / 1.30 / 0.033 / 549 / 40 / 8
4 / O75347 / Tubulin-specific chaperone A / TBCA / 1.06 / 1.63 / 1.54 / 0.009 / 460 / 36 / 13
11 / P14618 / Pyruvate kinase isozyme M1/M2 / PKM / 1.06 / 0.81 / -1.31 / 0.028 / 1216 / 44 / 21
15 / P04406 / Glyceraldehyde 3-phosphate dehydrogenase / GAPDH / 1.12 / 0.84 / -1.33 / 0.035 / 486 / 50 / 13
20 / P04406 / Glyceraldehyde 3-phosphate dehydrogenase / GAPDH / 1.13 / 0.81 / -1.39 / 0.048 / 1035 / 59 / 18
16 / no I.D. / 1.10 / 0.80 / -1.38 / 0.038
6 / P27797 / Calreticulin / CALR / 1.25 / 0.90 / -1.39 / 0.016 / 1681 / 43 / 25
8 / P22695 / Cytochrome b-c1 complex subunit 2, mitochondrial / UQCRC2 / 1.07 / 0.77 / -1.39 / 0.020 / 565 / 31 / 9
14 / P10768 / S-formylglutathione hydrolase (esterase D) / ESD / 1.08 / 0.76 / -1.42 / 0.035 / 268 / 24 / 8
17 / O43707 / Alpha actinin-4 / ACTN4 / 1.22 / 0.78 / -1.56 / 0.041 / 1069 / 28 / 21
12 / P07355 / Annexin A2 / ANXA2 / 1.14 / 0.70 / -1.63 / 0.030 / 2856 / 69 / 89
2 / P00558 / Phosphoglycerate kinase 1 / PGK1 / 1.16 / 0.74 / -1.64 / 0.003 / 481 / 43 / 16
10 / P00558 / Phosphoglycerate kinase 1 / PGK1 / 0.17 / 0.80 / -1.47 / 0.021 / 943 / 51 / 30
7 / A8K486 / Peptidyl- cis-trans isomerase / PPIA / 1.14 / 0.76 / -1.49 / 0.018 / 506 / 50 / 17
9 / A8K486 / Peptidyl- cis-trans isomerase / PPIA / 1.40 / 0.77 / -1.82 / 0.021 / 733 / 50 / 27
18 / A8K486 / Peptidyl- cis-trans isomerase / PPIA / 1.07 / 0.80 / -1.33 / 0.041 / 348 / 43 / 13
a Spot numbers refer to those in Figure S1.
b Accession numbers of proteins were derived from the Swiss-Prot database.
c The normalized abundance was calculated by dividing the average spot intensity of a Cy3 or Cy5 labelled sample by the average spot intensity of the Cy2 labelled internal standard.
d Fold-difference (FD) was calculated by dividing the normalized abundance of a protein in the poor prognosis group by that for good prognosis.
e p-values were calculating using 1-Way ANOVA.
PP, poor prognosis; GP, good prognosis. Calreticulin (bold) was verified by SRM.
Table S4. Summary of iTRAQ 2DLC-MS/MS analyses of lymph node metastases from poor (n = 14, < 1 year survival) and good (n = 19, > 4 years survival) prognosis melanoma patients
Set 1 / 1583 / 18797 / 57997 / 79.3
Set 2 / 1271 / 14655 / 37171 / 76.8
Set 3 / 1431 / 16215 / 50176 / 79.8
Set 4 / 1228 / 16175 / 41509 / 80.4
Set 5 / 1120 / 11896 / 29187 / 79.4
Set 6 / 1051 / 11639 / 33828 / 78.8
Table S5. Reproducibility of iTRAQ 2DLC-MS/MS experiments as assessed using Pearson’s correlation of log2 transformed iTRAQ 113 labelled internal control data (Median Pearson's coefficient = 0.76)
Set 1-113 / Set 2-113 / Set 3-113 / Set 4-113 / Set 5-113 / Set 6-113Set 1-113 / 1.00 / 0.72 / 0.82 / 0.71 / 0.72 / 0.66
Set 2-113 / 0.72 / 1.00 / 0.74 / 0.83 / 0.86 / 0.79
Set 3-113 / 0.82 / 0.74 / 1.00 / 0.81 / 0.73 / 0.73
Set 4-113 / 0.71 / 0.83 / 0.81 / 1.00 / 0.87 / 0.76
Set 5-113 / 0.72 / 0.86 / 0.73 / 0.87 / 1.00 / 0.79
Set 6-113 / 0.66 / 0.79 / 0.73 / 0.76 / 0.79 / 1.00
Table S7. Differentially abundant proteins between good and poor prognosis AJCC stage IIIc melanoma patients identified using iTRAQ and 2DLC-MS/MS
noa / Protein name / Gene
name / Pept.
no.b / FDc
PP/GP / Adj.
p-valued / mRNA FDe
PP/GP
A8K402 / T-complex protein 1 subunit beta / CCT2 / 1 / 2.60 / 0.08 / n/a
P12956 / X-ray repair cross-complementing protein 6, Ku antigen, 70 kDa / XRCC6 / 18 / 2.44 / 0.01 / -1.04
P02452 / Collagen alpha-1(I) chain / COL1A1 / 4 / 2.03 / 0.03 / -1.10
P08238 / Heat shock protein HSP 90-beta / HSP90AB1 / 25 / 1.95 / 0.00 / 1.20
P02790 / Hemopexin / HPX / 8 / 1.91 / 0.01 / n/a
P60842 / Eukaryotic initiation factor 4A-I / EIF4A1 / 6 / 1.90 / 0.00 / -1.08
Q92820 / Gamma-glutamyl hydrolase / GGH / 3 / 1.80 / 0.05 / 1.26
P23526 / Adenosylhomocysteinase / AHCY / 8 / 1.75 / 0.01 / 1.07
O75431 / Metaxin 2 / MTX2 / 2 / 1.68 / 0.04 / 1.27
P09874 / Poly [ADP-ribose] polymerase 1 / PARP1 / 10 / 1.67 / 0.00 / 1.07
P30046 / D-dopachrome decarboxylase / DDT / 4 / 1.66 / 0.03 / 1.22
P01009 / Alpha-1-antitrypsin / SERPINA1 / 29 / 1.63 / 0.04 / -1.60
P01024 / Complement C3 / C3 / 37 / 1.63 / 0.00 / -1.16
P00738 / Haptoglobin / HP / 17 / 1.63 / 0.01 / -1.06
Q99798 / Aconitate hydratase, mitochondrial / ACO2 / 11 / 1.63 / 0.05 / 1.09
P51884 / Lumican / LUM / 12 / 1.60 / 0.06 / -1.92
Q6IBN1 / Heterogeneous nuclear ribonucleoprotein K / HNRNPK / 14 / 1.59 / 0.00 / n/a
Q15233 / Non-POU domain-containing octamer-binding protein / NONO / 7 / 1.59 / 0.00 / 1.13
P02787 / Serotransferrin / TF / 33 / 1.59 / 0.00 / 1.21
P10644 / cAMP-dependent protein kinase type I-alpha regulatory subunit / PRKAR1A / 6 / 1.58 / 0.02 / -1.10
P13010 / X-ray repair cross-complementing protein 5, Ku antigen, 80 kDa / XRCC5 / 15 / 1.58 / 0.01 / -1.04
P35580 / Myosin-10 / MYH10 / 6 / 1.56 / 0.02 / 1.31
P23396 / 40S ribosomal protein S3 / RPS3 / 12 / 1.55 / 0.00 / -1.24
P00338 / L-lactate dehydrogenase A chain / LDHA / 22 / 1.49 / 0.00 / -1.04
Q99832 / T-complex protein 1 subunit eta / CCT7 / 10 / 1.48 / 0.03 / 1.10
Q08211 / ATP-dependent RNA helicase A / DHX9 / 12 / 1.48 / 0.04 / 1.02
P62826 / GTP-binding nuclear protein Ran / RAN / 7 / 1.46 / 0.01 / -1.08
P22087 / rRNA 2'-O-methyltransferase fibrillarin / FBL / 6 / 1.44 / 0.05 / 1.07
P07237 / Protein disulfide-isomerase / P4HB / 32 / 1.41 / 0.00 / 1.06
Q00839 / Heterogeneous nuclear ribonucleoprotein U / HNRNPU / 20 / 1.37 / 0.01 / 1.05
P11142 / Heat shock cognate 71 kDa protein / HSPA8 / 26 / 1.34 / 0.08 / -1.29
P40926 / Malate dehydrogenase, mitochondrial / MDH2 / 17 / 1.31 / 0.08 / 1.18
Q9Y3E5 / Peptidyl-tRNA hydrolase 2, mitochondrial / PTRH2 / 1 / 1.30 / 0.06 / 1.24
P07195 / L-lactate dehydrogenase B chain / LDHB / 15 / 1.25 / 0.08 / 1.07
P14314 / Glucosidase 2 subunit beta / PRKCSH / 14 / -1.32 / 0.07 / 1.10
P27797 / Calreticulin / CALR / 25 / -1.36 / 0.02 / -1.06
Q96C19 / EF-hand domain-containing protein D2 / EFHD2 / 5 / -1.37 / 0.08 / -1.40
B1AKZ3 / Phosphoprotein enriched in astrocytes 15 (PEA15) / PEA15 / 3 / -1.37 / 0.07 / n/a
Q99497 / Protein DJ-1 (Oncogene DJ1) / PARK7 / 14 / -1.38 / 0.04 / -1.33
P78417 / Glutathione S-transferase omega-1 / GSTO1 / 15 / -1.40 / 0.08 / -1.06
P26038 / Moesin / MSN / 20 / -1.42 / 0.01 / -1.21
P30048 / Thioredoxin-dependent peroxide reductase, mitochondrial / PRDX3 / 8 / -1.42 / 0.09 / -1.10
P04083 / Annexin A1 / ANXA1 / 22 / -1.49 / 0.00 / -1.41
Q09666 / Neuroblast differentiation-associated protein AHNAK / AHNAK / 93 / -1.49 / 0.00 / -1.16
P53999 / Activated RNA polymerase II transcriptional coactivator p15 (p14) / SUB1 / 8 / -1.51 / 0.05 / -1.07
Table S7: continued