Supplementary Information
NF-κB Expression and Outcomes in Solid Tumors: A Systematic Review and Meta-Analysis
Dang Wu, Pin Wu, Lufeng Zhao, Lijian Huang, Zhigang Zhang, Shuai Zhao and Jian Huang *
Supplementary data：
Figure S1. Subgroup analysis of three-year overall survival (OS) by Nuclear factor-kappaB (NF-κB)
expression in different tumor types.
Figure S2. Subgroup analysis of Nuclear factor-kappaB (NF-κB) expression in different TNM stages of
tumor.
Figure S3. Subgroup analysis of three-year overall survival (OS) by cellular localization of Nuclear
factor-kappaB (NF-κB) expression. 1 = Nuclear expression; 2 = Cytoplasmic expression.
Figure S4. Subgroup analysis of five-year overall survival (OS) by Nuclear factor-kappaB (NF-κB)
expression in different tumor types.
Figure S5. Subgroup analysis of five-year overall survival (OS) by cellular localization of Nuclear
factor-kappaB (NF-κB) expression. 1 = Nuclear expression; 2 = Cytoplasmic expression.
Table S1. Evaluation of human NF-kB by immunohistochemistry (IHC) in the selected studies
NF-kB
Antibody
References
Country
Type of cancer
Marker
(+)%
(Clone)
Abdel-Latif, M.
Esophageal
NF-kB
M., et al.
USA
60.82%
NRd
carcinoma
p65-N/Ca
(2004)
Annunziata, C.
NF-kB
M., et al.
Norway
Ovarian cancer
58.06%
sc-1190
p50-N/C
(2010)
Head and Neck
Balermpas, P.,
NF-kB
Germany
Squamous Cell
33.66%
ab31481
et al. (2013)
p65-Nb
Carcinoma
Berardi, R., et
NF-kB
Italy
Rectal cancer
47.95%
NR
al. (2012)
p65-N
Chu, D., et al.
(2011)
China
Colorectal cancer
NF-kB
p65-N/C
48.85%
ab7970
Darb-Esfahani,
S., et al. (2010)
Germany
Ovarian cancer
NF-kB
p65-Cc
55.29%
sc8008
Method and Cut-off for overexpression
NR
Positive: IHC >25% tumor cells were
positive for NF-kB staining.
Positive: IHC ≥5% tumor cells were
positive for NF-kB staining.
Positive: IHC ≥1% tumor cells were
positive for NF-kB staining.
IHC, score ≥5. The extensional standards
taken were as follows: (i) number of
positive stained cell 5%, scored 0;
6%–25%, scored 1; 26%–50%, scored 2;
51%–75%, scored 3; and >75%, scored 4
and (ii) intensity of stain: colorless,
scored 0; pallideflavens, scored 1; yellow,
scored 2; and brown scored 3.
The extensional standards (i) and (ii)
weremultiplied and the staining grade was
stratified as absent (0 score), weak (1–4
score), moderate (5–8 score), and strong
(9–12 score).Tumors with moderate or
strong immunostaining were classified as
having positive expression.
IHC, IRS >4. Total cytoplasmic p65
expression was grouped as followed:
“negative”, IRS ≤4; “positive”, IRS >4.
Guo, R. X., et
al. (2008)
Hatata, T., et al.
(2012)
Huang, C., et
al. (2009)
Izzo, J. G., et al.
(2007)
Izzo, J.
G.,Correa, A.
M., et al.
(2006)
Izzo, J.
G.,Malhotra,
U., et al. (2006)
Jiang, L. Z., et
al. (2011)
Jin, X., et al.
(2008)
Kleinberg, L.,
et al. (2009)
Korkolopoulou
, P., et al.
(2008)
Kwon, H. C., et
al. (2010)
Kwon, H. C., et
al. (2012)
Lee, B. L., et al.
(2005)
Levidou, G., et
al. (2007)
Esophageal
carcinoma
Laryngeal squamous
cell carcinoma
Esophageal
carcinoma
NF-kB
p65-N/C
NF-kB
p65-N/C
NF-kB
p65-N/C
NF-kB
p65-N
USA
Esophageal
carcinoma
NF-kB
p65-N
58.75%
G96-337
Positive: IHC ≥5% tumor cells were
positive for NF-kB staining.
USA
Esophageal
carcinoma
NF-kB
p65-N
48.84%
G96-337
Positive: IHC ≥5% tumor cells were
positive for NF-kB staining.
Laryngeal squamous
cell carcinoma
Non-small cell lung
cancer
NF-kB
p65-N/C
NF-kB
p65-N/C
NF-kB
p65-N/C
64.04%
sc502
46.59%
sc109
66.18%
NR
96.34%
sc114
47.30%
NR
42.61%
NR
17.59%
NR
91.40%
sc114
China
Japan
China
USA
China
China
Ovarian cancer
Norway
Ovarian cancer
Greece
Astrocytomas
Korea
Colorectal cancer
Korea
Gastric cancer
Korea
Gastric cancer
Greece
Gastric cancer
NF-kB
p50-N/C
NF-kB
p65-C
NF-kB
p65-C
NF-kB
p65-N
NF-kB
p50-C
75.00%
NR
59.42%
sc109
60.26%
NR
64.23%
G96-337
Positive: IHC >50% tumor cells were
positive for NF-kB staining.
Positive: IHC ≥10% tumor cells were
positive for NF-kB staining.
Positive: IHC ≥1% tumor cells were
positive for NF-kB staining.
Positive: IHC ≥5% tumor cells were
positive for NF-kB staining.
Positive: IHC ≥10% tumor cells were
positive for NF-kB staining.
Positive: IHC >5% tumor cells were
positive for NF-kB staining.
Positive: IHC >25% tumor cells were
positive for NF-kB staining.
Positive: IHC ≥15% tumor cells were
positive for NF-kB staining.
Positive: IHC >10% tumor cells were
positive for NF-kB staining.
Positive: IHC >10% tumor cells were
positive for NF-kB staining.
Positive: IHC ≥10% tumor cells were
positive for NF-kB staining.
Positive: IHC ≥10% tumor cells were
positive for NF-kB staining.
Lewander, A.,
et al. (2012)-1b
Lewander, A.,
et al. (2012)-2c
Li, J., et al.
(2009)-M1
Li, J., et al.
(2009)-M2
Lin, G., et al.
(2012)
Nair, V. S., et
al. (2014)
Nariai, Y., et al.
(2011)
O'Neil, B. H., et
al. (2011)
Sweden
Colorectal cancer
Sweden
Colorectal cancer
China
Cervical cancer
China
Cervical cancer
China
Colorectal cancer
USA
Japan
USA
Non-small-cell lung
cancer
Oral squamous cell
carcinoma
Rectal cancer
p-NF-kB
p65-N
p-NF-kB
p65-C
NF-kB
p65-N
NF-kB
p50-N
NF-kB
p65-N/C
NF-kB
p65-C
NF-kB
p65-N/C
NF-kB
p50-N
36.45%
NR
64.04%
NR
63.29%
sc372
82.28%
sc114
30.56%
NR
57.19%
D14E12
52.00%
sc8008
92.06%
sc114
Okera, M., et
al. (2011)
USA
Prostate cancer
NF-kB
p65-N/C
52.73%
Cat#RB-163
8
Pancione, M.,
et al. (2009)
Italy
Colorectal cancer
NF-kB
p65-N/C
58.33%
sc109
Positive: IHC ≥5% tumor cells were
positive for NF-kB staining.
Positive: IHC ≥5% tumor cells were
positive for NF-kB staining.
Positive: IHC ≥10% tumor cells were
positive for NF-kB staining.
Positive: IHC ≥10% tumor cells were
positive for NF-kB staining.
NR.
Positive: IHC >20% tumor cells were
positive for NF-kB staining.
Positive: IHC >81% tumor cells were
positive for NF-kB staining.
IHC, >180 units.
IHC, score ≥3. A numeric intensity score of
0–3 was assigned to each case (0,
no staining; 1+, weak staining; 2+,
moderate staining and 3+, strong staining).
Staining was also dichotomized into
negative and positive. Negative was scored
if 0+/1+ and positive was scored if 2+/3+.
IHC, score ≥3. The intensity was scored as
follows: 1 (weak), 2 (moderate), or 3
(strong). The percentage of positive cells
was scored as follows: 0 (0%), 1
(1%-25%), 2 (26%-50%), 3 (51%-75%), or
4 (76%-100%). The 2 scores were
combined to obtain the final one: negative
(0-2), weakly positive (3-5), or strongly
positive (6-7).
Park, K. W., et
al. (2014)
Scartozzi, M.,
et al. (2007)
Sun, W., et al.
(2012)
Voboril, R., et
al. (2008)
Korea
Gastric cancer
Italy
Colorectal cancer
China
Nasopharyngeal
carcinoma
Czech
Republic
Rectal cancer
NF-kB
p65-C
NF-kB
p65-N/C
NF-kB
p65-N/C
NF-kB
p65-C
44.16%
NR
Positive: IHC >10% tumor cells were
positive for NF-kB staining.
60.53%
NR
NR.
55.45%
sc109
NR.
20.00%
NR
IHC, Presence of NF-kB staining.
Weichert, W.,
et al. (2007)-1
Germany
Pancreatic cancer
NF-kB
p65-N
45.12%
sc8008
Weichert, W., et
al. (2007)-2
Germany
Pancreatic cancer
NF-kB
p65-C
51.22%
sc8008
IHC, IRS ≥7. Intensity of staining was
designated as either not existent (0),
weak (1), moderate (2) or strong (3). The
number of cells stained was scored
as either no cells stained (0), less than 10%
of cells stained (1), 10– 50% of cells
stained (2), 50– 80% of cells stained (3) or
more than 80% of cells stained (4).
The IRS was calculated by multiplication of
these two variables.
For statistical analysis, cases were grouped
as either negative (IRS 0 –6)
or positive (IRS 7 –12).
IHC, IRS ≥7. Intensity of staining was
designated as either not existent (0),
weak (1), moderate (2) or strong (3). The
number of cells stained was scored
as either no cells stained (0), less than 10%
of cells stained (1), 10– 50% of cells
stained (2), 50– 80% of cells stained (3) or
more than 80% of cells stained (4).
The IRS was calculated by multiplication of
these two variables.
For statistical analysis, cases were grouped
Wu, W., et al.
(2008)
Wu, Z., et al.
(2013)
Yamanaka, N.,
et al. (2004)
Yang, G., et al.
(2011)
Yeh, H. C., et
al. (2010)
Yoon, S. O., et
al. (2009)
Voboril, R., et
al. (2008)
Zhang, D., et al.
(2007)
China
Gallbladder
carcinoma
China
Cervical cancer
Japan
Gastric cancer
USA
Ovarian cancer
NF-kB
p65-N/C
NF-kB
p65-N/C
NF-kB
p65-N
NF-kB
p65-N
57.89%
NR
72.50%
NR
33.33%
sc109
80.56%
NR
as either negative (IRS 0 –6)
or positive (IRS 7 –12).
Positive: IHC >10% tumor cells were
positive for NF-kB staining.
Positive: IHC ≥10% tumor cells were
positive for NF-kB staining.
Positive: IHC >25% tumor cells were
positive for NF-kB staining.
NR.
IHC, score ≥5. The intensity of cytoplasmic
staining was graded by a score of
1 point (weak), 2 points (moderate), and 3
points intense). The percentage of
stained cells was rated as 1 point (≤10%), 2
points (11–50%), and 3 points
(>50%). Cases with a sum of ntensity points
and percentage points of ≥5 were
defined as having an overexpression of
NF-κB.
Positive: IHC >10% tumor cells were
positive for NF-kB staining.
China
Urothelial carcinoma
NF-kB
p65-N
26.67%
sc109
Korea
Mucinous
adenocarcinoma
NF-kB-N
40.00%
NR
Czech
Republic
Rectal cancer
21.74%
NR
NR
China
Non-small cell lung
cancer
48.28%
NR
Positive: IHC >5% tumor cells were
positive for NF-kB staining.
NF-kB
p65-C
NF-kB
p65-N/C
Zhang, J., et al.
(2005)
China
Adenoid Cystic
Carcinoma of
Salivary Glands
NF-kB
p65-N
Zhang, Y., et al.
China
(2011)
Nasopharyngeal
carcinoma
NF-kB
p65-N
Zhang, Z., et al.
China
(2006)
Zhou, Q., et al. China
Non-small cell lung
cancer
Intrahepatic
NF-kB
p50-N/C
NF-kB-N/
35.00%
NR
64.29%
NR
66.67%
NR
51.52%
NR
IHC, score ≥4. The intensity of staining was
on the following scale: 0, no
staining; 1+, mild staining; 2+, moderate
staining; 3+, intense staining. The area
of staining was evaluated as follows: 0, no
staining of cells in any microscopic
fields; 1+, <25% of tissue stained positive;
2+, between 25% and 50% stained
positive; 3+, between 50% and 75% stained
positive; 4+, >75% stained positive.
The minimum score when summed
(extension + intensity) was therefore 0 and
the maximum, 7. The combined staining
score (extension + intensity) V3 was
considered as negative staining (low
staining); a score between 4 and 5 was
considered as moderate staining; between 6
and 7 was strong staining.
IHC, score ≥4. The score is the sum of the
percentage of positive cells (1, less
than 25% positive cells; 2, 26 to 50%
positive cells; and 3, more than 50%
positive cells) and the staining intensity (0,
negative; 1, weak; 2, moderate; 3,
strong). Sums between 0 and 3 were scored
as negative; sums from 4 to 6 were
scored as positive.
Positive: IHC ≥10% tumor cells were
positive for NF-kB staining.
IHC, score ≥3. First, the intensities of
(2013)
Cholangiocarcinoma
C
staining were indicated as follows: 0, no
staining; 1, light yellow; 2, brownish
yellow; 3, reddish brown. Second, relative
numbers of positive cells were marked as
follows: 0, no positive cells; 1, positive
cells ≤ 10%; 2, positive cells 11–50%; 3,
positive cells 51–75%; 4, positive cells
>75%. Finally, all sections were divided
into four grades by the product of
intensity scores and percentage scores
described above: –, 0–2 points; +, 3–4
points; 2+, 6–8 points; 3+, 9–12 points; ≥ 3
points defined as positive.
a. N/C: Nuclear or cytoplasmic expression; b. N,1: Nuclear expression; c. C,2: Cytoplasmic expression; d. NR: No report.