ARTICLE
Auteur(s) : Xiaoli Feng1, Ling Li1,
Yanning Gao2, Jianjun Zhang2, Jianming
Ying1, Ting Xiao2, Jidong Gao3,
Xiuyun Liu1, Yuntian Sun1, Shujun
Cheng2
1Department of pathology, Cancer Hospital and
Institute, Chinese Academy of Medical Sciences, Beijing 100021
2Department of etiology, Cancer Hospital and Institute,
Chinese Academy of Medical Sciences, Beijing 100021
3Department of abdomenal surgery, Cancer Hospital and
Institute, Chinese Academy of Medical Sciences, Beijing 100021
Fragile histidine triad (Fhit) is a non classical tumour suppressor
gene. Abnormalities in this gene are among the most common genetic
changes occurring in human cancers [1, 2]. But the significance of
Fhit protein reduction in lung cancer requires additional studies.
This study was performed for the purpose of using one kind of new
technology (tissue microarray, TMA) and the immunohistochemistry
(IHC) method to examine Fhit protein expression its significance in
the development or progression in lung cancer in a Chinese
population.
Material and methods
A total of 321 lung cancer cases were obtained from patients who
underwent surgical resection at the Cancer Hospital (Chinese
Academy of Medical Science, CAMS.) between 1990 and 1993.
Furthermore 101 metastasized lymph nodes corresponding with these
tumors were also assessed.
Reagents used included Fhit multi-clone antibody (dilution 1 in
25, Zymed, USA), the ultra sensitive SP kit (Fuzhou Maixin
biotechnology CO, LTD) and DAB (diaminobenzidine
tetrahydrochloride) detection kit (Zhongshan Golden Bridge
Biotechnology CO, LTD).
The construction of microarray was used by a punching manual
operation machine (Beecher Instruments arraying device, USA). This
kind of instrument is designed to produce sample spots that are
0.6 mm in diameter at a spacing of 1.0 mm. First we
collected the part of the cases that contained the cancer required
for the array by using a microscope and reviewing the slides and
matching the blocks. We circled the area of tumour present in the
slide. This was the area that was biopsied (cored) and placed in
the recipient block forming the array. An array block was 27 x 22 x
12 mm. The cores were placed in the block (according to
redundancy) to fill this area. We established two lung cancer
microarray blocks altogether 435 samples, including 321 tumors, in
which 150 squamous cell carcinomas (SCC), 127 adenocarcinomas (Ad),
18 adeno-squamous carcinomas (AdSq) and 26 small cell lung cancers
(SCLC). Moreover 101 metastasized lymph nodes corresponding to the
primary tumours and 13 normal lung tissues (n = 7) and lymph nodes
(n = 6) were studied as internal controls. Sections of
5 μm-thickness were cut from paraffin-embedded tissues. Each
10 slides we stained a HE slice (figures 1 à 4).
Sections of 5 μm-thickness were deparaffinated in xylene
and rehydrated in a graded series of ethanol. Immunohistochemistry
was performed according to the SP method. Negative controls were
carried out by omitting the primary antibody instead of PBS.
External positive controls used external controls from tumors well
known to be positive for Fhit antibody. We stained three TMA slices
continuously. Immunostaining was classified into the following four
groups: [1] negative, no staining was present, or positive staining
was detected in < 5 %; [2] positive staining ranging from 6
to 25 % of cell stained; [3] positive staining ranging from 26
to 50 % of cell stained; [4] positive immunostaining was
present in > 50 % of the cells. Observers (n = 2)
independently evaluated the results of the immunohistochemical
staining without any knowledge of the clinical data. Positive cells
for Fhit were cytoplasmic brown granular staining.
Statistical analysis: Basic data was processed using software
SPSS10.0 on computer. The different rate comparison used
χ2 examination to carry on the analysis. Survival
distribution was estimated by the Kaplan-Meier method. The
criterion for statistical significance was p < 0.05.
Results
The histological subtypes of patients included 150 SCCs, 18 AdSq,
127 Ads and 26 SCLC basis on 1999 WHO lung and pleural membrane
tumor classification. The mean age was 56 years (ranging from 29 to
83).Male and female patients were 243 and 78 respectively. The male
and female proportion was 3.1:1. The three grading of SCCs and Ads
consisted of 15 low grade [1], 92 intermediate [2], 43 high grade
[3] in SCC and 30 low grade [1], 49 intermediate [2], 48 high grade
[3] in Ad. 157 patients had stage I disease, 86 stage II, 78 stage
III according to the 1997 UICC. The median follow-up was 59 months
(range 0-118).
The number of tumour specimens assessable for Fhit protein
expression were 253 (78.8 %). Among the 101 patients
simultaneously having the primary tumours and the corresponding
metastasized lymph nodes, 54 (53.5 %) were assessable for FHIT
expression in both samples. Finally the concordance rate of TMA
slice staining and the conventional slice staining was bigger than
93 %.
The expression rate of the Fhit protein (intermediate to strong
intensity (++, +++)) was 32.9 % (83/253). The remaining 170
(67.1 %) patients were characterized by completely absence of
cytoplasmic staining or marked reduction of immunoreactivity.
Non-tumorous components (normal bronchial epithelial cells,the
bronchial gland and II pulmonary alveolus epithelium) were internal
Fhit positive controls. The expression of Fhit protein was stronger
in NSCLC than in SCLC (p < 0.05).A loss and reduction of Fhit
protein was statistically significant between patients with smoking
(133/191,69.6 %)and without smoking (29/62, 46.8 %) (p
< 0.005).The difference expression of Fhit protein were observed
in males (135/189, 71.4 %) and females (29/64, 45.3 %) (p
< 0.005). The loss and reduction of Fhit protein was not
significantly associated with age, stage, presence of lymph node
metastasis or survival time (table 1,
table 2), (figure 5).
Table 1 The expression of Fhit protein with relation to
clinical and pathological parameters
|
Parameters
|
No. of patients (%)
|
Fhit
|
|
|
- (%)
|
+ (%)
|
++ (%)
|
+++ (%)
|
|
Stage
|
|
|
|
|
|
|
I
|
120 (47.4)
|
44 (36.7)
|
34 (28.3)
|
25 (20.8)
|
17 (14.2)
|
|
II
|
65 (25.7)
|
23 (35.4)
|
30 (32.3)
|
13 (20.0)
|
8 (12.3)
|
|
III
|
68 (26.9)
|
29 (42.6)
|
28 (27.9)
|
14 (20.6)
|
6 (8.9)
|
|
ARRAY(0x208d64)
|
|
Histological subtype
|
|
|
|
|
|
|
SCC
|
105 (41.5)
|
45 (42.9)
|
40 (38.1)
|
20 (19.0)
|
0 (0)
|
|
Ad
|
109 (43.1)
|
23 (21.1)
|
30 (27.5)
|
27 (24.8)
|
29 (26.6)
|
|
AdSq
|
18 (7.1)
|
11 (61.1)
|
3 (16.7)
|
2 (11.1)
|
2 (11.1)
|
|
SCLC
|
21 (8.3)
|
17 (81.0)
|
1 (4.8)
|
3 (14.2)
|
0 (0)
|
|
ARRAY(0x20c19c)
|
|
Grade (SSC and Ad)
|
|
|
|
|
|
|
1
|
39 (18.2)
|
17 (43.6)
|
9 (23.1)
|
8 (20.5)
|
5 (12.8)
|
|
2
|
104 (48.6)
|
24 (23.0)
|
40 (38.5)
|
23 (22.2)
|
17 (16.3)
|
|
3
|
71 (33.2)
|
27 (38.0)
|
21 (29.5)
|
16 (22.5)
|
7 (10.0)
|
|
ARRAY(0x20f2a4)
|
|
Sex
|
|
|
|
|
|
|
male
|
189 (74.7)
|
135 (71.4%)a
|
54 (28.6)b
|
|
female
|
64 (25.3)
|
29 (45.3)a
|
35 (54.7)b
|
|
ARRAY(0x212214)
|
|
Smoke
|
|
|
|
|
|
|
positive
|
191 (75.5)
|
133 (69.6%)a
|
58 (30.4)b
|
|
negative
|
62 (24.5)
|
29 (46.8)a
|
33 (53.2)b
|
aFhit expression frequency - ~ + (%).
bFhit expression frequency ++ ~ +++ (%).
Table 2 The expression of Fhit protein with the
relation to the primary tumors and metastasized lymph nodes
|
Fhit protein expression in metastasized lymph nodes
|
No. of patients (%)
|
Fhit protein expression in the primary tumors
|
|
- (%)
|
+ (%)
|
++ (%)
|
+++ (%)
|
|
-
|
16 (29.6)
|
12 (75.0)
|
2 (12.4)
|
1 (6.3)
|
1 (6.3)
|
|
+
|
23 (42.6)
|
4 (17.4)
|
13 (56.5)
|
4 (17.4)
|
2 (8.7)
|
|
++
|
9 (16.7)
|
1 (11.1)
|
3 (33.3)
|
3 (33.3)
|
2 (22.3)
|
|
+++
|
6 (11.1)
|
0 (0)
|
1 (16.7)
|
2 (33.3)
|
3 (50.0)
|
Discussion
We studied Fhit protein expression using two lung cancer TMAs
through IHC method, and stained three TMA slices continuously. This
may increase the number of assessable specimens. We also confirmed
in 10 cases that TMA tissue preparations were coherent with
conventional slice staining. In the literature researchers also
confirmed TMA technical reliability [8-10]. Although the TMA
operation of initial period of choosing representative region was
quite time-consuming, and had some difficulties in later period of
reading slides, there are numerous advantages to this technology
including amplification of a scarce resource; experimental
uniformity; not destroying original block for diagnosis; greatly
saving the manpower, physical resource and time comparing with the
traditional convention slice staining. It may speed up the
application step of the molecular markers in the clinical diagnosis
and treatment.
The Fhit gene, located at 3p14.2, is one kind of candidate
suppressor genes encompassing the most fragile site of the human
genome, FRA3B. Loss of this gene are among the most common genetic
changes occurring in lung cancer. Recently studies showed that the
change of Fhit through DNA or RT-PCR analysis showed Fhit loss in
lung, uterine cervix and breast cancer [3-5]. These results
displayed the change of Fhit gene was very easily to be examined
through the IHC analysis. The method had the superiority in the
examination of protein loss.
There are very few reports exploring FHIT expression in Chinese
patients with lung cancer. In this study the assessable number of
tumor specimens was 253 (78.8 %). The total loss and the
obvious decline (-, +) rate of Fhit protein in lung cancer was
67.1 % (37.9 and 29.2 %). The rate of SCC, AdSq, Ad and
SCLC respectively were 81.0 % (42.9 and 38.1 %),
77.8 % (61.1 and 16.7 %), 48.6 (21.1 and 27.5 %) and
85.8 % (81.0 and 4.8 %).
Our results of reduced Fhit protein expression mostly concerned
cases of SCLCs and SCCs, but not Ads. Moreover the obvious drop of
Fhit gene protein expression often occurred in patients who smoked,
but not in non-smokers, in keeping with previous reports [6, 7].
Our results suggest that abnormal Fhit protein expression may be
involved in tumorigenesis of SCLC and SCC, as was suggested by
other authors [8, 1, 2].
The relation between Fhit gene and prognosis, in our conclusion,
was that loss or decline of Fhit protein expression was not
correlated with prognosis. These results are similar to those of
Sozzi [4] and Tseng [9]. The conclusion of Tomizawa was opposite
[2].
The reduced expression was often observed in the less
differentiated 2 and 3 grade of lung SCCs and Ads, especially in
the Ads, as in the report by Chang et al. [8]. In the literature,
Fhit protein expression was reported to be correlated with ethnic
origin.Our study also suggested a correlation between Fhit protein
expression and male sex. A possible explanation of this phenomenon
was the fact that most male patients were smokers, while few female
patients were smokers.
In summary, TMA technology greatly saves manpower, physical
resources and time. It was advantageous in the high throughput
analysis. It is good in performing translational research of new
molecular markers. The loss or decline of Fhit protein is frequent
in lung SCCs and SCLCs. Its role in the occurrence and outcome of
patients with lung cancer will require the analysis of larger
series.
Acknowledgement
This study was supported by Chinese National Nature Science
Foundation (No.30270582).
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