PINCH is an independent prognostic factor in rectal cancer patients without preoperative radiotherapy--a study in a Swedish rectal cancer trial of preoperative radiotherapy.

Abstract

The clinical significance between particularly interesting new cysteine-histidine rich protein (PINCH) expression and radiotherapy (RT) in tumours is not known. In this study, the expression of PINCH and its relationship to RT, clinical, pathological and biological factors were studied in rectal cancer patients.
PINCH expression determined by immunohistochemistry was analysed at the invasive margin and inner tumour area in 137 primary rectal adenocarcinomas (72 cases without RT and 65 cases with RT). PINCH expression in colon fibroblast cell line (CCD-18 Co) was determined by western blot.
In patients without RT, strong PINCH expression at the invasive margin of primary tumours was related to worse survival, compared to patients with weak expression, independent of TNM stage and differentiation (P = 0.03). No survival relationship in patients with RT was observed (P = 0.64). Comparing the non-RT with RT subgroup, there was no difference in PINCH expression in primary tumours (invasive margin (P = 0.68)/inner tumour area (P = 0.49). In patients with RT, strong PINCH expression was related to a higher grade of LVD (lymphatic vessel density) (P = 0.01)
PINCH expression at the invasive margin was an independent prognostic factor in patients without RT. RT does not seem to directly affect the PINCH expression.

Full text

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RESEARCH ARTICLEOpen Access
PINCH is an independent prognostic factor in
rectal cancer patients without preoperative
radiotherapy - a study in a Swedish rectal cancer
trial of preoperative radiotherapy
Annica Holmqvist1*, Jingfang Gao1, Birgitta Holmlund1, Gunnar Adell1, John Carstensen2, Dianne Langford3and
Xiao-Feng Sun1
Abstract
Background: The clinical significance between particularly interesting new cysteine-histidine rich protein (PINCH)
expression and radiotherapy (RT) in tumours is not known. In this study, the expression of PINCH and its
relationship to RT, clinical, pathological and biological factors were studied in rectal cancer patients.
Methods: PINCH expression determined by immunohistochemistry was analysed at the invasive margin and inner
tumour area in 137 primary rectal adenocarcinomas (72 cases without RT and 65 cases with RT). PINCH expression
in colon fibroblast cell line (CCD-18 Co) was determined by western blot.
Results: In patients without RT, strong PINCH expression at the invasive margin of primary tumours was related to
worse survival, compared to patients with weak expression, independent of TNM stage and differentiation (P =
0.03). No survival relationship in patients with RT was observed (P = 0.64). Comparing the non-RT with RT
subgroup, there was no difference in PINCH expression in primary tumours (invasive margin (P = 0.68)/inner
tumour area (P = 0.49). In patients with RT, strong PINCH expression was related to a higher grade of LVD
(lymphatic vessel density) (P = 0.01)
Conclusions: PINCH expression at the invasive margin was an independent prognostic factor in patients without
RT. RT does not seem to directly affect the PINCH expression.
Keywords: PINCH, Radiotherapy, Prognosis, Rectal cancer, Immunohistochemistry
Background
Particularly interesting new cysteine-histidine rich protein
(PINCH) is a five LIM domain protein whose gene is
located on chromosome 2q12.2. PINCH is a part of the
PINCH-ILK-Parvin (PIP) complex connected to integrins
at the cell surface, and acts as an adapter protein for signal
transduction through the cytosol [1]. The PIP complexes
provide crucial physical linkages between integrins and the
actin cytoskeleton and transduce signals from the extracel-
lular matrix to intracellular effectors [2,3]. These effectors
further regulate the cytoskeleton organisation, spreading,
motility and proliferation of the cell [4,5]. PINCH is up-
regulated in several types of cancers, and increases from
normal mucosa to primary tumour to metastasis [6-9]. In
previous studies of colorectal tumours, PINCH was more
intensely stained at the invasive margin compared to the
intratumoural stroma, and related to worse prognosis
[6,7].
Few have analysed the relationship between PINCH
and radiotherapy (RT). Previously, it was shown that
PINCH was radio-resistant by activating Akt [10].
Others found PINCH to be similarly radio-sensitive
under adherent and suspension conditions [11]. RT is
known to regulate the cell cycle in the G1, G2and S-
phase and PINCH has been shown to be involved in cell
cycle progression and survival [4,5,12]. Since both
* Correspondence: annica.holmqvist@lio.se
1Department of Medical Oncology, Institute of Clinical and Experimental
Medicine, Linköping University, S-58185 Linköping, Sweden
Full list of author information is available at the end of the article
Holmqvist et al. BMC Cancer 2012, 12:65
http://www.biomedcentral.com/1471-2407/12/65
© 2012 Holmqvist et al; BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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PINCH and RT seems to be involved in cell cycle regu-
lation and the location of PINCH at the invasive margin
seems to play an important role in patient’s prognosis,
we wanted to investigate the relationship between
PINCH and RT, both at the invasive margin and inner
tumour area, in primary rectal tumours.
The aim of this study was to investigate PINCH
expression in tumours and its relationship to RT, clini-
cal (gender, age, TNM stage, differentiation, local recur-
rence, distant recurrence and survival), pathological
(lymph-angiogenesis, angiogenesis, inflammatory infiltra-
tion and necrosis) and biological factors (apoptosis) in
rectal cancer patients with or without RT.
Immunohistochemical studies of colorectal tumours
have shown that PINCH was widely expressed in the
stroma around tumour cells [6,7]. Here, cell lines of
normal fibroblasts were used to study the expression of
PINCH and to further analyse the relationship between
PINCH expression and RT.
Methods
Patients
This study included patients from the Southeast Swedish
Health Care region who participated in a Swedish clinical
trial of preoperative RT during 1987-1990 [13]. All
patients were diagnosed with rectal adenocarcinoma. The
present study included 137 primary tumours, where 72
patients underwent tumour resection alone and 65
patients underwent preoperative RT before surgery. RT
was administered with 25 Gray (Gy) in 5 fractions during
a median of 6 days (range, 5-12 days). Surgery was then
performed a median of 3 days (range, 1-13 days) after RT.
None of the patients received adjuvant chemotherapy
before or after surgery. The mean age of the patients was
67 years (range, 36-85 years) and the median follow
up was 86 months (range, 0-193 months). Additional char-
acteristics of the patients and tumours are present in
Table 1. The Research Ethics Committee professor Åke
Bertler at Linkoping University hospital, number 86151,
approved the study.
The level of lymphangiogenesis and angiogenesis [14]
were determined by immunohistochemistry. Inflamma-
tory infiltration and necrosis were analysed on HE-
stained sections [15] and apoptotic cells were detected by
the terminal deoxynucleotidy transferase-mediated
dUTP-biotin nick end-labelling (TUNEL) assay [16]. The
data were taken from our previous studies performed at
our laboratory.
Immunohistochemistry
Five-micrometer formalin fixed, paraffin-embedded sec-
tions were deparaffinised in xylene, rehydrated with a
graded series of ethanol to water. The sections were
treated by high pressure cooking for 10 min with Tris-
ethylenediaminetetraacetic acid (EDTA) buffer (pH 9.0)
and kept at room temperature for 30 min. Following
pre-incubation in methanol with 0.3% H2O2for 20 min,
the sections were incubated with protein block (Dako,
Carpinteria, CA) for 10 min and then incubated with
rabbit anti-PINCH antibody at 6 μg/ml in antibody dilu-
ent (Dako) for 1 h at room temperature. After washing
in phosphate-buffered saline (PBS, pH 7.4), the sections
were incubated with an anti-rabbit/mouse secondary
antibody provided by Dako ChemMate EnVision Detec-
tion Kit (Dako) at room temperature for 25 min and
washed with PBS. Subsequently, the sections were sub-
jected to 3,3’-diaminobenzidine tetrahydrochloride for 8
min and then counterstained with hematoxylin. The
positive controls were primary colorectal tumours
known to stain positive for PINCH and the negative
controls were primary rectal tumours where PBS was
used instead of the primary antibody. In all staining pro-
cedures, the positive controls showed clear immunos-
taining but no immunostaining was observed in the
negative controls.
Table 1 Patient and tumour characteristic (n = 137)
Characteristics
Non-RT n (%) RT n (%)
P
Gender
Male 42 (58)40 (62)0.70
Female30 (42)25 (38)
Age (years)0.84
≤ 6730 (42)26 (42)
> 6742 (58)39 (60)
TNM0.11
I 20 (28)22 (34)
IIA 18 (25)21 (32)
IIIA8 (11) 1 (2)
IIIB 11 (15)11 (17)
IIIC 11 (15)4 (6)
IV4 (6)6 (9)
Differentiation0.63
Good2 (3)2 (3)
Moderate58 (81) 48 (74)
Poor 12 (16)15 (23)
Surgical type0.17
Rectal amputation 36 (50)25 (38)
Anterior resection36 (50) 40 (62)
Resection margin 0.33
Tumor free 70 (97) 61 (94)
Tumor 2 (3)4 (6)
To anal verge (cm)
Mean 7.58.5
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The staining results of PINCH in tumours were the
mean of scores by two independent authors (A. Holmq-
vist and J. Gao) in a blinded fashion without any knowl-
edge of clinical and biological information.
The staining intensity was determined in 10-20 areas
(depending on the size of the section) at 400 × magnifi-
cation. The cases were considered to have negative,
weak, moderate or strong staining. The percentage of
stained cells was estimated among the total number of
cells by reading 10-20 areas at 400 × magnification,
regardless of the staining intensity. The cases were
scored as < 25%, 25-49%, 50-75%, or > 75%, respectively.
To avoid artificial effect, the cells on the margins of sec-
tions and areas with poorly presented morphology were
not counted. In the cases with discrepant results in the
staining score, a consensus score was reached after re-
examination.
Cell culture and radiation procedure
The CCD-18 Co cell line derived from human colon
fibroblasts (ATCC, Rockville, MD), was a kind gift from
Dr. R Palmqvist (Department of Pathology, Umeå Uni-
versity, Sweden). The cells were cultured in Dulbecco’s
Modified Eagles Medium (DMEM) with Glutamax™
and supplemented with 1% Penicillin-Streptomycin and
10% FBS (Invitrogen, Carlsbad, CA).
For all experiments, cells were seeded at a density of
60.000 cells/cm2and irradiated with photons from a 6 MV
linear accelerator Varian Clinac 600 C/D (Varian Medical
Systems, Palo Alto, CA). The field size was 30 × 30 cm
and the distance between sources and cells was 100 cm.
Acrylic glass plates were placed above (3 cm thick) and
underneath (10 cm thick) the cells.
The cells were exposed to single doses of 0, 2, 5 or 10
Gy at room temperature. The most significant biological
change in protein expression was observed with the radia-
tion dose of 2 Gy, as also shown by previous studies [17].
Therefore 2 Gy was used for further analyses in our study.
The controls (0 Gy) were handled under the same envir-
onmental conditions as the treated cells. Following radia-
tion, cells were harvested at 8, 24, 48 and 72 h for western
blot analysis. All experiments were repeated three times.
Western blot analysis
After radiation, cells where washed in PBS and lysed in
RIPA buffer, containing 150 mM NaCl 2% Triton, 0.1%
SDS, 50 mM Tris pH 8.0 and a Protease Inhibitor Cocktail
without chelating reagents (Sigma-Aldrich, Stockholm,
Sweden). Protein concentration was determined with the
colorimetric BCA protein assay reagent (Pierce, Woburn,
MA). Samples containing 30 μg protein where separated
by electrophoresis in a Mini-PROTEAN TGX™ precast
12% Gel (Bio-Rad, Hercules, CA) for 55 min at 200 V. The
separated proteins were transferred to a PVDF-membrane
(Amersham Biosience/GE Healthcare, Piscataway, NJ).
The membranes were blocked with 5% non-fat dried milk
in Tris-buffered saline (TBS) containing 0.1% Tween 20
(TBST) and incubated with primary PINCH antibody
(REF) 1 μg/mL overnight at 4°C in TBST and 1% non-fat
dried milk. The membranes were washed and incubated
for 1 h at room temperature with a HRP conjugated poly-
clonal goat anti-mouse secondary antibody (1:5,000, Dako,
Cytomation, Glostrup, Denmark) followed by enhanced
chemiluminescence (ECL)(Amersham Biosiences/GE
Healthcare). To verify equal loading of the wells the mem-
branes were reincubated with a primary mouse polyclonal
anti-b-actin antibody (1:5,000, Sigma-Aldrich, Steinheim,
Germany) and a secondary polyclonal goat ant-mouse
antibody (1:10,000, Dako, Cytomation, Glostrup,
Denmark).
Statistical analysis
The Chi-square method was used to analyse the relation-
ship between PINCH expression in tumours and the clini-
cal, pathological or biological factors. Cox’s proportional
hazard model was used to estimate the relationship
between PINCH expression and survival, including both
univariate and multivariate analyses. Survival curves were
computed according to the Kaplan-Meier method. Tests
were two-sided and P <0.05 was considered statistically
significant.
Results
PINCH expression in primary tumours
PINCH protein expression was analysed at the invasive
margin (Figure 1A) and inner tumor area (Figure 1B)
of 137 primary tumours. At the invasive margin 16
(12%) cases had weak PINCH expression, 51 (37%)
cases had moderate and 70 (51%) cases had strong
PINCH expression. At the inner tumour area 12 (9%)
cases had weak expression, 66 (48%) cases moderate
and 59 (43%) cases had strong PINCH expression.
There were no negative cases for PINCH either at the
invasive margin or inner tumour area. In this study, no
statistically significant results where found when the
percentage of stained cells where analysed (P > 0.05),
therefore, further analysis only shows the result of the
staining intensity.
Staining scores for PINCH in primary tumours with and
without RT are presented in Table 2. For further analysis
the expression levels of PINCH were classified in either a
weakly stained subgroup if they were scored as negative,
weak or moderately stained, or into a strong subgroup if
they were strongly stained (Table 2).
Upon comparing the PINCH expression at the inva-
sive margin with that at the inner tumour area in the
137 primary tumours, 23 of the cases (17%) had stron-
ger staining at the invasive margin, 99 of the cases
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(72%) showed the same staining levels, and 15 cases
(11%) had weaker staining (P = 0.06).
PINCH expression in primary tumours in relation to
clinical variables
We further analysed the relationships between PINCH
expression at both the invasive margin and at the inner
tumour area of primary tumours with patient survival.
At the invasive margin of tumours, either in all patients
(P = 0.04) or in the non-RT group (P = 0.03, Figure
2A), strong expression of PINCH was related to shorter
survival time, compared to those with weak PINCH
expression. In the non-RT group, a further multivariate
analysis showed that the prognostic significance still

A
B
Figure 1 Strong PINCH expression in tumour-associated stroma at the invasive margin (A) and inner tumour area (B) of primary rectal
tumours.
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remained after adjusting for both TNM stage and differ-
entiation (P = 0.03). In patients with RT, no significant
difference was found between the expression of PINCH
and survival time (P = 0.64, Figure 2B). A further inter-
action analysis between PINCH (at the invasive margin),
RT and survival did not show a statistically significant
result (P = 0.30).
When the inner tumour area was analysed, no signifi-
cant relationship was present for PINCH expression and
survival in patients from the non-RT or RT subgroups
(P > 0.05).
No significant differences were found between the
subgroups of non-RT and RT in PINCH expression of
either at the invasive margin (P = 0.68) or inner tumor
area (P = 0.49) as shown in Table 2.
There was no significant relationship of PINCH
expression of either at the invasive margin or inner
tumour area with local recurrence, distant recurrence or
disease free survival, in the whole group of patients and
in the subgroups of non-RT and RT in primary tumours
(p > 0.05).
PINCH expression in primary tumours in relation to
pathological and biological factors
We further analysed PINCH expression of primary
tumours at the invasive margin and at the inner tumour
area and the relationship to clinical, pathological and
biological factors.
In all patients, strong PINCH expression was related
to weak inflammatory infiltration (P = 0.002) and a
higher grade of necrosis (P = 0.03) at the invasive mar-
gin of tumours. In the non-RT subgroup, strong PINCH
expression was related to weak inflammatory infiltration
(73% of 33 cases Vs 37% of 35, P = 0.003) and less
apoptosis (66% of 35 cases Vs 36% of 33, P = 0.02) and
positively related to age (P = 0.005). There was no sig-
nificant relationship in the RT group (P > 0.05).
In all patients and in the non-RT subgroup, strong
PINCH expression at the inner tumour area was related
to a higher blood vessel density (BVD) located at the
periphery (P = 0.03, P = 0.02) and weak inflammatory
infiltration (P = 0.0005, P = 0.0007). In the RT group,
strong PINCH expression was related to a higher grade
of LVD (P = 0.01, Figure 3) and more necrosis (P =
0.01).
PINCH expression in CCD-18 co with or without RT
The protein expression of PINCH in CCD-18 Co cells
was studied without (0 Gy) and with RT (2 Gy), and
analysed over time at 8, 24, 48 and 72 h after RT. The
expression of PINCH in the CCD-18 Co cells showed
equally thick single clear bands as shown in Figure 4.
No differences were observed in PINCH protein expres-
sion between cells without RT (-, 0 Gy) or with RT (+,
2 Gy) harvested at different times.
Discussion
This is the first study of PINCH expression in rectal
cancer patients who participated in a Swedish clinical
trial of preoperative RT. In patients without RT, strong
PINCH expression at the invasive margin of primary
tumours was related to worse survival compared to
patients with weak PINCH expression. The prognostic
significance still remained even after adjustment for
both TNM stage and differentiation. This result was in
line with others who studied PINCH expression in 174
colorectal cancer patients [7]. After RT, there was no
relationship between PINCH and survival. A further
interaction analysis showed no statistically significant
result, which might indicate that the number of deaths
in the RT group was low. As far as we know, this is the
first study of PINCH in relation to RT in patients. A
previous cell line study of mouse embryonic fibroblasts
and human colon, lung, cervix, skin and pancreas
tumours showed that PINCH was radio-resistant by
activating Akt1 [10]. Others showed that the radio sensi-
tivity in PINCH depleted normal and malignant cells
was similar under adherent and suspension conditions
[11]. In this study, we did not found any significant dif-
ference in PINCH expression between the subgroups of
non-RT and RT, which might be explained by a too
short interval between RT and surgery. The best clinical
effects of RT on tumour tissue are known to be received
around 5 weeks after RT. In our study, the patients
received preoperative RT and went through surgery
within 1-13 days after RT, which might be a too short
time to receive the optimal clinical effect by RT. Even
though there was no significant difference in PINCH
expression between the non-RT and RT subgroups, the
survival for patients with weak and strong PINCH
expression seemed to change with RT, which makes us
suggest that PINCH might not be directly increased by
RT, but maybe activated by RT via other biological
pathways.
Table 2 PINCH expression in primary rectal tumours with
or without RT
Staining intensity
Weak n (%) Strong n (%)
P
Invasive margin
Non-RT34 (47)38 (53)
RT 33 (51)32 (49)
P = 0.49
Inner tumour area
Non-RT 39 (54)33 (46)
RT39 (60)26 (40)
P = 0.68
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