Supplementary Table S1 – Scoring of C4.4A levels

Supplementary Table S1 – Scoring of C4.4A levels

Histology Variable N^a Median Range

Squamous cell carcinoma

Center 102 8 0-16

Periphery 100 8 0-14

Adenocarcinoma Center 84 1 0-16

Periphery 82 2 0-14

Adenocarcinoma growth patterns

Mucinous lepidic Center 6 0 0-3

Periphery 5 0 0-3

Non-mucinous lepidic

Center 8 0 0-0

Periphery 8 0 0-6

Acinar Center 30 0 0-12

Periphery 31 0 0-12

Micropapillary Center 10 0 0-6

Periphery 7 0 0-2

Papillary Center 13 0 0-9

Periphery 15 0 0-4

Solid Center 50 4 0-16

Periphery 44 4 0-16

a

N for growth patterns indicates the number of patients where the given pattern represents at least

10% of the tumor tissue examined.

Supplementary Table S2 – Analysis of overall survival (squamous cell carcinoma)

Univariate Multivariate

Covariate Hazard ratio

(95% CI)

p-value Hazard ratio (95%

CI)

p-value

Stage IIA+B vs. stage IA+B

Stage IIIA vs. stage IA+B

2.01 (0.97-4.15)

3.17 (1.44-6.99)

0.017

2.92 (1.24-6.87)

4.32 (1.67-11.16)

0.009

Performance status (1+2 vs. 0) 1.11 (0.61-2.02) 0.74 0.62^b

Gender (Female vs. Male) 0.65 (0.26-1.65) 0.37 0.60^b

Age (pr. 10 years) 0.89 (0.59-1.34) 0.57 0.87^b

Treatment (Adjuvant vs. none^a) 0.69 (0.26-1.83) 0.45 0.45^b

C4.4A center score (pr. 4 units) 1.08 (0.78-1.49) 0.64 0.82^b

C4.4A periphery score (pr. 4 units) 1.06 (0.73-1.52) 0.77 0.83^b

a

For stages II and III only and adjusted for stage

b

P-value to include covariate in the model

The analysis was performed on patients in stages I through IIIA, thus excluding 9 patients in stages

IIIB and IV.

Supplementary Table S3: Scoring of C4.4A levels according to adenocarcinoma growth patterns

C4.4A immunohistochemical expression in the different growth patterns of adenocarcinomas (mucinous and non-mucinous lepidic, acinar, papillary, micropapillary, and solid) was analyzed semi-quantitatively by scoring the intensity and frequency of C4.4A positivity in tumor cells separately from 0 to 4, yielding final scores in the range 0-16. For most patients, two sections (A & B) were examined.

Patient # Section Mucinous lepidic Non-mucinous lepidic Acinar Papillary Micropapillary Solid Predominant pattern

1 A 0

B 0 0 Papillary

2 A 0 0 0 Acinar

3 A 3 9 Solid

B 6 9 Solid

4 A 4

B 0 2 Acinar

5 A 12 4 Acinar

B 2 6 9 Acinar

6 A 12

B 9

7 A 0

B 3

8 A 2 3 Solid

B 4 4 Solid

9 A 0 0 Papillary

B 0 0 0 Papillary

10 A 0 1 1 Papillary

B 0

11 A 0 2

B 3

12 A 1 12 Non-m lepidic

B 2 2 6 Lepidic

13 A 16

B 16

14 A 0 0 Acinar

B 0 2 Acinar

15 A 0 0 50-50 acinar-solid

16 A 0 0 Micropapillary

17 A 6

B 6

18 A 0 0 Acinar

B 0 0 Acinar

19 A 0 0 Acinar

B 0

20 A 2 0 Acinar

B 2 0 Acinar

21 A 6 9 Solid

B 12 Solid

22 A 0 8 Papillary

B 3 6 Papillary

23 A 0

B 0

24 A 4

B 4

25 A 0 0 Papillary

B 6 0 Acinar

26 A 4

B 6 12 Papillary

27 A 0 2 Solid

B 0 1 Solid

28 A 9 2 Solid

B 2

29 A 9

B 9

30 A 2

B 6 0 3 Acinar

31 A 2 8 Solid

B 0 3 Solid

32 A 0

B 0 4 8 Acinar

33 A 9

B 12

34 A 6 6 Acinar

35 A 1 2 3 Solid

B 2 9 Acinar

36 A 0

B 0

37 A 4

B 4

38 A 0 6 Acinar

B 0 0 Acinar

39 A 0

B 0

40 A 0

B 0

41 A 3 16 Acinar

B 3 16 Acinar

42 A 3 4 Solid

B 3 4 Solid

43 A 9

B 9

44 A 0

45 A 0 0 0 Acinar

B 0 0 0 Acinar

46 A 0 0 Solid

B 0 0 Solid

47 A 12

B 12

48 A 12

B 12

49 A 3 12 Muc lepidic

B 3 4 8 Muc lepidic

50 A 3 3 Acinar

B 2 0 9 Acinar

51 A 0 0 Solid

B 0 0 Solid

52 A 0

B 0

53 A 4 16 Papillary

B 9

54 A 4

B 4

55 A 0 0 Acinar

B 0 0 0 Acinar

56 A 0 0 Acinar

B 0 0 Acinar

57 A 1 Acinar

B 0 1 Acinar

58 A 0 0 Non-m lepidic

59 A 0 3 Solid

B 0 4 Solid

60 A 4 4 Solid

B 0 1 Solid

61 A 16

B 12

62 A 0 6 Solid

B 0 4 Solid

63 A 12

64 A 6

B 6

65 A 6 6 Solid

B 6 6 Solid

66 A 0 0 Solid

B 0 0 Solid

67 A 0 0 Solid

B 0 0 Solid

68 A 2 2 Solid

B 0 2 Solid

69 A 4

B 3

70 A 0 0 Micropapillary

B 0 0 Micropapillary

71 A 0

B 0 0

B 0

73 A 0

74 A 0 0 Solid

B 0 1 Solid

75 A 16

76 A 4

B 4

77 A 6

B 6

78 A 6 12 Solid

B 6 12 Solid

79 A 0 0 Acinar

B 0 0 Acinar

80 A 6

B 3

81 A 0 6 Solid

B 6

82 A 12 16 Solid

B 12 16 Solid

83 A 0 4 0 12 Acinar

84 A 3

B 3

85 A 0 0 Acinar

B 0 0 Acinar

86 A 0 2 Solid

B 0 4 Solid

87 A 0

B

C

D

E

C'

D'

E' A

Figure 1 supp

Supplementary Figure S1

Staining specificity of the anti-C4.4A polyclonal antibody

Several experiments controlling for the reproducibility and specificity of the polyclonal antibody were performed to make sure that the semi-quantitative evaluation of C4.4A expression levels would be unbiased. Positive controls included staining of the esophagus (panel A) and the amnion membrane of human term placenta (panel B). At the gastro-esophageal junction present in the probed esophagus sections (panel A), the prominent C4.4A expression in the squamous esophageal epithelium disappeared upon transition to the columnar gastric epithelium.

Panels C, D, and E depict NSCLC specimens from the present patient material probed for C4.4A.

Adjacent sections of these cases were either challenged with pre-absorption of the polyclonal

antibody by a 10-fold molar excess of recombinant C4.4A protein before addition to the tissue

(panel C'), with an irrelevant rabbit IgG (panel D'), or by omitting the 1° antibody in the staining

procedure (panel E'). All these controls eliminated the reactivity for C4.4A, in line with earlier

specificity tests for the anti-C4.4A polyclonal antibody in archival formalin-fixed and paraffin-

embedded samples of pulmonary tissue (Jacobsen B et al, Int J Cancer, 2012). Scale bars: 100 µm

(A, C-E’); 50 µm (B).