# Stat3 Expression and Its Correlation with Proliferation and Apoptosis/Autophagy in Gliomas

Authors: Valentina Caldera; Marta Mellai; Laura Annovazzi; Guido Valente; Luciana Tessitore; Davide Schiffer
Journal: Journal of Oncology
 (2008)

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## Abstract

Signal transducer and activator of transcription-3 (Stat3) was studied along with several steps of the PI3/Akt pathway in a series of 64 gliomas that included both malignant and low-grade tumors, using quantitative immunohistochemistry, Western blotting, and molecular biology techniques. The goal of the study was to investigate whether activated Stat3 (phospho-Stat3) levels correlated with cell proliferation, apoptosis, and autophagy. Stat3 and activated Akt (phospho-Akt) expression increased with malignancy grade, but did not correlate with proliferation and survival within the category of glioblastomas. A correlation of Stat3 with Akt was found, indicating a regulation of the former by the PI3/Akt pathway, which, in turn, was in relation with EGFR amplification. Stat3 and Akt did not show any correlation with apoptosis, whereas they showed an inverse correlation with Beclin 1, a stimulator of autophagy, which was rarely positive in glioblastomas. Autophagy seems then to be inactivated in malignant gliomas.


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## Body

## 1. Introduction

Stat3 is a member of a family of latent transcription factors that transduce
extra-cellular signals, such as those
mediated by cytokines and growth factors [1]. Stat3 is activated by the phosphorylation of a tyrosine residue
(phospho-Stat3), which leads to its dimerization and nuclear translocation.
It transcriptionally regulates
the expression of genes responsible for proliferation and survival. pStat3 has been shown to suppress apoptosis in many cancers [2, 3].pStat3 is
ubiquitously expressed in mammalian cells and is constitutively activated in
glioblastomas by IL-6 [4]. It has been shown that in many cancers when pStat3
is inhibited, its target genes, Bcl-XL, c-myc, and Cyclin D1, are down-regulated,
and cells undergo apoptosis [3, 5, 6].pStat3 is
not detected in normal brain tissue. It is
activated by aberrant EGFR signaling and IL-6 in malignant gliomas [7], and it
may be a rational therapeutic target; WP10066, similar to AG490, has been shown
to inhibit pStat3 and to induce apoptosis in vitro and in vivo [8].
A pStat3 mutant can induce cellular transformation and tumor formation in nude
mice by binding DNA and activating transcription [5], and inhibition of pStat3
may promote the efficacy of immunotherapy [9]. In a series of diffuse gliomas,
pStat3 has been found to be focally expressed in less than 9% of gliomas [10].
However, in another series, it was highly expressed at almost equal levels in
anaplastic astrocytomas and glioblastomas (55.6% versus 56.4%), together with pAkt,
and its expression also correlated with EGFR
status (EGFRvIII), but had no predictive value [11]. pStat3
was found to be positive in 40% of gliomas and correlated with histological
grades [12]. Recently, it has been demonstrated that both inhibitors of the
PI3/Akt pathway [13] and pStat3 can induce autophagy in glioma cells [14].Autophagy is a caspase-independent process of
degradation, characterized by the formation of autophagosomes and their fusion
with lysosomes [15]. It is regulated by pmTOR and its complexes [16], by
autophagy genes (ATG) and Beclin 1 [17]. Beclin 1 is also known as Atg6, and is
a component of a complex that includes the class III
phosphatidylinositol-3-kinase and is an autophagic stimulator [16].Quantitative
evaluation of pStat3 expression in gliomas is difficult due to the regional heterogeneity of its expression. We
investigated pStat3 expression and its
correlations with histological grade, EGFR aberrations,
PTEN mutations, pAkt expression, cell proliferation, and apoptosis/autophagy in a series of gliomas.


## 2. Materials and Methods

Surgical
samples were collected from the Department of Neuroscience, University of Turin,
and from the Clinical and Experimental Medical Department of East Piedmont, University of Novara. Sixty four gliomas were studied:
34 glioblastomas (GBMs), 10 grade III anaplastic astrocytomas, 10 grade II
astrocytomas, and 10 oligodendrogliomas (5 grade II and 5 grade III), diagnosed
according to the WHO.All the
tumor samples were from first surgery with partial or total removal. No
recurrence and no second-operation materials were studied.Surgical
samples were fixed in buffered formalin, embedded in paraffin and cut in 5-micron thick serial sections.


### 2.1. DNA Extraction

Genomic DNA was extracted
from the 34 formalin-fixed, paraffin-embedded GBM samples according to a
standard phenol-chlorophorm protocol. Only proliferating areas were selected.


### 2.2. EGFR (Epidermal Growth Factor Receptor) Amplification

EGFR amplification status was assessed as previously
described [18]. Fluorescent PCR products were analyzed by capillary
electrophoresis on an ABI
PRISM 3100 Genetic Analyzer (Applied Biosystems,
Foster City, CA, USA).
The amplification status of the EGFR gene was determined by measuring the
EGFR/INF-γ ratio: a ratio >2.09 was
taken as evidence of more than two copies of the EGFR gene.


### 2.3. PTEN (Phosphatase and Tensin Homolog) Mutation Analysis

The PTEN gene was amplified as ten polymerase chain
reaction (PCR) fragments covering the 9 exons and at least 50 bp of flanking intronic sequence.All the fragments were amplified
using the same protocol; an initial denaturation at 96°C for 10 minutes followed by 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds, for 33 cycles. A final elongation
step of 10 minutes at 72°C was
added. The reactions were performed in a total volume of 25μL containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 250 μM of each dNTP, 1 unit of Taq Gold polymerase (Applied Biosystems), 10 pmol of each primer and 100 ng of genomic DNA. Primer sequences
are available upon request.Prior to sequencing, unincorporated dNTPs and primers
were removed by using MultiScreen PCR filter plates on a MultiScreenHTS vacuum manifold (Millipore, MA Billerica, USA).Each PCR product was further analyzed by direct DNA
sequencing in both directions on an ABI
PRISM 3100 Genetic Analyzer using the
BigDye Terminator version 1.1 cycle sequencing Kit.


### 2.4. Immunohistochemistry (IHC)

The
following primary antibodies were used. Mouse monoclonal antiphospho-Stat3
(Tyr705), diluted 1:40 (#9138, Cell Signaling Technology, Beverly, MA, USA);
mouse monoclonal antiphospho-Akt (Ser473), diluted 1:100 (#4051, Cell
Signaling Technology); rabbit polyclonal anti-EGFRwt, diluted 1:50 (#2232,
Cell Signaling Technology); rabbit polyclonal anti-EGFRvIII, diluted 1:50
(SC1031, GenScript Corporation, Piscataway, NJ, USA); mouse monoclonal
anti-PTEN (A2B1), diluted 1:1000 (sc-7974, Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse monoclonal anti-Ki-67/MIB.1, diluted 1:100 (M7240, Dako,
Carpinteria, CA, USA); rabbit polyclonal anti-Caspase-3, diluted 1:20 (AB3623,
Chemicon International Inc., Temecula, CA, USA); rabbit polyclonal anti-PARP1,
diluted 1:200 (#9542, Cell Signaling Technology); rabbit polyclonal
anticleaved PARP1 (Asp214), diluted 1:50 (#9541, Cell Signaling Technology), rabbit
polyclonal anti-Beclin 1, diluted 1:200 (sc-11417, Santa Cruz Biotechnology).Besides the H&E method,
immunohistochemistry was performed on consecutive sections using a standard streptavidin-biotin
system (Dako) with diaminobenzidine as the substrate (DAB, Roche Diagnostics
GmbH, Penzberg, Germany), and the sections were counterstained with Harris’
hematoxylin. Antigen retrieval was performed by microwaving the sections in 0.01 M citrate buffer (pH
6.0 or 7.4) (3×3 minutes at 600 W). Negative controls were only incubated with
the secondary antibody. The positive controls were human breast cancer samples for
pStat3 and pAkt, malignant neuroblastoma for Caspase-3, PARP1 and cleaved-PARP1, and a
glioblastoma sample with no mutations for PTEN. A primary human glioblastoma
with EGFR gene amplification
was used as a positive control for the wild type EGFR (EGFRwt).


### 2.5. Evaluation of Immunohistochemical Staining

In all of the samples, only the proliferating areas
were evaluated, while necrotic or regressive areas
were excluded from analysis. Antigen
expression was evaluated according to the intensity (–, +, ++),
frequency of positive nuclei/cells (<20%, 20%–50%, >50%), and distribution (focal or diffuse),
with a scoring system that used three categories A, B, and C (Table1).Table 1
Scoring system for evaluation of immunostaining.
MarkerCategoryABCpStat3 and pAktDiffuse, <20%, + with or without fociDiffuse, 20–50%, ++ with or without fociDiffuse, >50% or >20%, +++
with
multiple fociKi-67/MIB.1<20%20–30%>30%EGFR AmplificationNon-amplifiedNon-amplifiedMore than 2 copiesEGFRwt−+/−+EGFRvIII−+/−+PTENPositive—NegativeDiffuse = homogeneous distribution of positive nuclei.Foci = circumscribed small areas with a higher percentage of positive nuclei.%  =  number of positive nuclei×100 nuclei.The
Labelling Index (LI) was calculated as the mean of the areas, of at least 1000
cells. Visual analysis was used to identify the areas with the highest
frequency of positive nuclei/cytoplasms. These areas were usually 5 HPF with
immersion oil, which corresponded to 0.001 mm2.In
GBMs only, Caspase-3 and cleaved-PARP1 expression was calculated as the
percentage of positive nuclei/cytoplasms after counting all of the tumor sections.In addition, in GBMs, the Ki-67/MIB.1 LI was
calculated also in areas that showed the maximum pStat3 LI, and the pStat3 LI was calculated in the areas with the maximum Ki-67/MIB.1
LI.


### 2.6. Protein Extraction and Western Blotting Analysis

Paraffin sections for protein extraction were
deparaffinized and homogenized in RIPA buffer with a
protease inhibitor cocktail (Sigma Aldrich Co., St. Louis,
MO, USA).
After quantification of the total protein lysate using the BCA Protein Assay
Kit (Pierce Biotechnology, Rockford,
IL, USA),
80μg of total
protein was resolved by SDS-PAGE on a 12% gel. The proteins were then
transferred to nitrocellulose membranes (Biorad, Hercules, CA, USA). Blots were incubated
overnight at 4°C with a rabbit monoclonal antiphospho-Akt (Ser473) antibody at
a dilution of 1:1000 (#3787, Cell Signaling Technology), and they were then
incubated with the appropriate HRP-conjugated secondary antibody (Dako).
Protein signals were detected using the ECL detection system (GE Healthcare, Buckinghamshire, UK). A specific anti-β-actin
antibody (A5441, Sigma Aldrich Co.) was used to normalize sample loading and
transfer. The intensity of the bands was quantified by densitometry using the
NIH Image J software (RSB, NIMH, Bethesda,
MD, USA).


### 2.7. Statistical Analysis

Associations between the variables were evaluated
using2×2 contingency tables and the two-tailed Fisher’s exact test. Correlation analyses
were performed using the Pearson’s correlation coefficient. Survival analysis
was carried out using the Kaplan-Meier method (SPSS version 15.0, Chicago, IL,
USA).


## 2.1. DNA Extraction

Genomic DNA was extracted
from the 34 formalin-fixed, paraffin-embedded GBM samples according to a
standard phenol-chlorophorm protocol. Only proliferating areas were selected.


## 2.2. EGFR (Epidermal Growth Factor Receptor) Amplification

EGFR amplification status was assessed as previously
described [18]. Fluorescent PCR products were analyzed by capillary
electrophoresis on an ABI
PRISM 3100 Genetic Analyzer (Applied Biosystems,
Foster City, CA, USA).
The amplification status of the EGFR gene was determined by measuring the
EGFR/INF-γ ratio: a ratio >2.09 was
taken as evidence of more than two copies of the EGFR gene.


## 2.3. PTEN (Phosphatase and Tensin Homolog) Mutation Analysis

The PTEN gene was amplified as ten polymerase chain
reaction (PCR) fragments covering the 9 exons and at least 50 bp of flanking intronic sequence.All the fragments were amplified
using the same protocol; an initial denaturation at 96°C for 10 minutes followed by 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds, for 33 cycles. A final elongation
step of 10 minutes at 72°C was
added. The reactions were performed in a total volume of 25μL containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 250 μM of each dNTP, 1 unit of Taq Gold polymerase (Applied Biosystems), 10 pmol of each primer and 100 ng of genomic DNA. Primer sequences
are available upon request.Prior to sequencing, unincorporated dNTPs and primers
were removed by using MultiScreen PCR filter plates on a MultiScreenHTS vacuum manifold (Millipore, MA Billerica, USA).Each PCR product was further analyzed by direct DNA
sequencing in both directions on an ABI
PRISM 3100 Genetic Analyzer using the
BigDye Terminator version 1.1 cycle sequencing Kit.


## 2.4. Immunohistochemistry (IHC)

The
following primary antibodies were used. Mouse monoclonal antiphospho-Stat3
(Tyr705), diluted 1:40 (#9138, Cell Signaling Technology, Beverly, MA, USA);
mouse monoclonal antiphospho-Akt (Ser473), diluted 1:100 (#4051, Cell
Signaling Technology); rabbit polyclonal anti-EGFRwt, diluted 1:50 (#2232,
Cell Signaling Technology); rabbit polyclonal anti-EGFRvIII, diluted 1:50
(SC1031, GenScript Corporation, Piscataway, NJ, USA); mouse monoclonal
anti-PTEN (A2B1), diluted 1:1000 (sc-7974, Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse monoclonal anti-Ki-67/MIB.1, diluted 1:100 (M7240, Dako,
Carpinteria, CA, USA); rabbit polyclonal anti-Caspase-3, diluted 1:20 (AB3623,
Chemicon International Inc., Temecula, CA, USA); rabbit polyclonal anti-PARP1,
diluted 1:200 (#9542, Cell Signaling Technology); rabbit polyclonal
anticleaved PARP1 (Asp214), diluted 1:50 (#9541, Cell Signaling Technology), rabbit
polyclonal anti-Beclin 1, diluted 1:200 (sc-11417, Santa Cruz Biotechnology).Besides the H&E method,
immunohistochemistry was performed on consecutive sections using a standard streptavidin-biotin
system (Dako) with diaminobenzidine as the substrate (DAB, Roche Diagnostics
GmbH, Penzberg, Germany), and the sections were counterstained with Harris’
hematoxylin. Antigen retrieval was performed by microwaving the sections in 0.01 M citrate buffer (pH
6.0 or 7.4) (3×3 minutes at 600 W). Negative controls were only incubated with
the secondary antibody. The positive controls were human breast cancer samples for
pStat3 and pAkt, malignant neuroblastoma for Caspase-3, PARP1 and cleaved-PARP1, and a
glioblastoma sample with no mutations for PTEN. A primary human glioblastoma
with EGFR gene amplification
was used as a positive control for the wild type EGFR (EGFRwt).


## 2.5. Evaluation of Immunohistochemical Staining

In all of the samples, only the proliferating areas
were evaluated, while necrotic or regressive areas
were excluded from analysis. Antigen
expression was evaluated according to the intensity (–, +, ++),
frequency of positive nuclei/cells (<20%, 20%–50%, >50%), and distribution (focal or diffuse),
with a scoring system that used three categories A, B, and C (Table1).Table 1
Scoring system for evaluation of immunostaining.
MarkerCategoryABCpStat3 and pAktDiffuse, <20%, + with or without fociDiffuse, 20–50%, ++ with or without fociDiffuse, >50% or >20%, +++
with
multiple fociKi-67/MIB.1<20%20–30%>30%EGFR AmplificationNon-amplifiedNon-amplifiedMore than 2 copiesEGFRwt−+/−+EGFRvIII−+/−+PTENPositive—NegativeDiffuse = homogeneous distribution of positive nuclei.Foci = circumscribed small areas with a higher percentage of positive nuclei.%  =  number of positive nuclei×100 nuclei.The
Labelling Index (LI) was calculated as the mean of the areas, of at least 1000
cells. Visual analysis was used to identify the areas with the highest
frequency of positive nuclei/cytoplasms. These areas were usually 5 HPF with
immersion oil, which corresponded to 0.001 mm2.In
GBMs only, Caspase-3 and cleaved-PARP1 expression was calculated as the
percentage of positive nuclei/cytoplasms after counting all of the tumor sections.In addition, in GBMs, the Ki-67/MIB.1 LI was
calculated also in areas that showed the maximum pStat3 LI, and the pStat3 LI was calculated in the areas with the maximum Ki-67/MIB.1
LI.


## 2.6. Protein Extraction and Western Blotting Analysis

Paraffin sections for protein extraction were
deparaffinized and homogenized in RIPA buffer with a
protease inhibitor cocktail (Sigma Aldrich Co., St. Louis,
MO, USA).
After quantification of the total protein lysate using the BCA Protein Assay
Kit (Pierce Biotechnology, Rockford,
IL, USA),
80μg of total
protein was resolved by SDS-PAGE on a 12% gel. The proteins were then
transferred to nitrocellulose membranes (Biorad, Hercules, CA, USA). Blots were incubated
overnight at 4°C with a rabbit monoclonal antiphospho-Akt (Ser473) antibody at
a dilution of 1:1000 (#3787, Cell Signaling Technology), and they were then
incubated with the appropriate HRP-conjugated secondary antibody (Dako).
Protein signals were detected using the ECL detection system (GE Healthcare, Buckinghamshire, UK). A specific anti-β-actin
antibody (A5441, Sigma Aldrich Co.) was used to normalize sample loading and
transfer. The intensity of the bands was quantified by densitometry using the
NIH Image J software (RSB, NIMH, Bethesda,
MD, USA).


## 2.7. Statistical Analysis

Associations between the variables were evaluated
using2×2 contingency tables and the two-tailed Fisher’s exact test. Correlation analyses
were performed using the Pearson’s correlation coefficient. Survival analysis
was carried out using the Kaplan-Meier method (SPSS version 15.0, Chicago, IL,
USA).


## 3. Results

### 3.1. EGFR Amplification and Immunohistochemistry (Performed Only in GBMs)

Twelve out of 34 GBMs showed EGFR amplification (35.3%), and 22 of the
GBMs were immunopositive for EGFRwt (64.7%). All but one of the 12 cases that
carried EGFR amplification were immunopositive for EGFRwt
(P  =  .239). Out
of the 22 cases that did not have EGFR amplification, 11 were immunopositive
for EGFRwt (score B  +  C).Out of the 12 cases with EGFR amplification, six were positive for
EGFRvIII (50%) (P  =  .0403), whereas out of the 22 cases without
amplification, only three were positive for EGFRvIII (13.6%) (Table 2).
Immunostaining for EGFRwt was either moderate or intense in most of the cells
analyzed (Figure 1(a)). Immunostaining for EGFRvIII was moderate, and showed
greater regional variability than the EGFRwt immunostaining (Figure 1(b)).Table 2
Correlation of EGFR gene
amplification status with EGFRwt and EGFRvIII immunohistochemistry in 34
glioblastomas.
EGFR amplificationEGFRwt (B  +  C) immunohistochemistryP valueEGFRvIII (B  +  C) immunohistochemistryP valuePositive (n=22)Negative (n=12)Positive (n=9)Negative (n=25)Positive12 (35.3%)11 (91.7%)1 (8.3%).02396 (50.0%)6
(50.0%).0403Negative22 (64.7%)11 (50.0%)11 (50.0%)3 (13.6%)19 (86.4%)(a)
Positive cells for EGFRwt, GBM. (b) Positive cells for EGFRvIII, GBM. (c) PTEN
positive staining, GBM. (d) Ki-67/MIB.1 high number of positive nuclei, GBM.
(e) pStat3 positive nuclei in GBM. (f) pStat3 negative nuclei in grade II
oligodendroglioma. DAB,400×.
(a)(b)(c)(d)(e)(f)Our cases of
glioblastoma were at their first surgery and none derived from a previous
astrocytoma. However, a distinction between primary and secondary glioblastoma
was not made, also because the only EGFR amplification is not an absolute
criterium.


### 3.2. PTEN Mutation Analysis and Immunohistochemistry (Performed Only in GBMs)

All the coding region and the intron-exon boundaries of the PTEN gene were amplified in
ten different PCR fragments in the 34 GBM cases.Direct sequencing led to the
identification of four different single nucleotide variations, located in the
coding region (n=1) and introns (n=3), respectively. These variations were
observed in the four patients resulted negative for PTEN expression by
immunohistochemistry.Variation in the coding region is a non-synonymous
substitution c.389G→A leading to an amino acid substitution at codon 130
(Arg130Gln), previously confirmed as somatic variant in high-grade gliomas and
in gliosarcomas [19, 20]. Among the three intronic variations, one is a new
sequence variation, namely IVS8+36C/T; the remaining were IVS8+32T/G and
IVS1−97A/G, corresponding to validated SNPs in public database of single nucleotide
polymorphisms (http://www.ncbi.nlm.nih.gov/SNP/) (rs555895 and rs1903858,
resp.) (Figure 2). The position of the intronic variations is relative
to the first (+1) or the last (−1) nucleotide of each intron (Gene Bank
sequence NM_000314).Nucleotide sequence analysis of PTEN exon 5. (a) The nonsynonymous substitution (Arg130Gln, CGA  >  CAA) from the tumor DNA of Case
6463. (b) The wild-type exon 5 sequence from normal control DNA.
(a)(b)Thirty out of the 34 GBMs were immunopositive for PTEN (88.2%). The
staining intensity was strong and uniform in all of the cases. Reactive
astrocytes were immunopositive for PTEN, and this staining was also seen in the
four tumors that were negative for PTEN immunostaining (Figure1(c)).


### 3.3. Ki-67/MIB.1

The LI was 5% (2–6%) for grade II astrocytomas, 12% (5–20%) for grade
III astrocytomas, 2% (0–10%) for grade II
oligodendrogliomas, 15% (12–28%) for grade
III oligodendrogliomas, and 23% (12–30%) for GBMs
(Figure1(d)).


### 3.4. Phospho-Stat3 Immunohistochemistry

pStat3 immunostaining was scored as “A” in almost all of the grade II
astrocytomas and oligodendrogliomas, as “B” in the grade III astrocytomas and
oligodendrogliomas, and as “B” or “C” in GBMs. The pStat3 staining was mainly
nuclear, with the exception of few scattered areas where it was cytoplasmic
(Figure1(e)). Cases with positivity <20%
were considered as negative. The percentages of positive cells were 0%, 50%,
and 56.2% for grade II, III astrocytomas and GBMs, respectively. In
oligodendrogliomas, the values were 0% and 20% for grade II and III,
respectively (Figure 1(f)).


### 3.5. Phospho-Akt Immunohistochemistry

In grade II astrocytomas and oligodendrogliomas, the staining was mostly
nuclear and was scored as “A”. In grade III astrocytomas and
oligodendrogliomas, the staining was still nuclear and was scored as “A”. In
GBMs, the staining was mainly cytoplasmic, and only occasionally nuclear, and
was scored as “C” (Figure3(a)). In GBMs
microvascular proliferations were negative and occasionally the staining was
more intense in cells around vessels and outside pseudopalisades. The frequency
values were 0%, 20%, and 80% for grade II, III astrocytomas and GBMs,
respectively. In oligodendrogliomas, the values were 0% and 20% for grade II
and III, respectively (Figure 3(b)).(a) Positive cytoplasmic staining for pAkt, GBM. (b)
Positive nuclear staining for pAkt in grade III oligodendroglioma. (c)
Caspase-3 positive nucleus, GBM. (d) Beclin 1 positive cytoplasmic and nuclear
staining, GBM. (e) PARP1 positive nuclei, GBM. (f) Cleaved-PARP1 positive
nucleus, GBM. DAB,400×.
(a)(b)(c)(d)(e)(f)


### 3.6. Phospho-Akt Western Blotting (Performed Only in GBMs)

The Western blotting analysis showed
variably positive bands (Figures4(a)-4(b)).(a) Western blotting of pAkt expression. Positive
bands in a sample of 8 cases. (b) Quantitative analysis of pAkt levels normalized toβ-actin data.
(a)(b)


### 3.7. Caspase-3, PARP1, and Cleaved-PARP1 Immunohistochemistry (Performed Only in GBMs)

Caspase-3 was positive as cytoplasmic
or nuclear staining or in apoptotic bodies (Figure3(c)). PARP1 was positive in all
the nuclei of the tumor (Figure 3(e)). Cleaved-PARP1 was
occasionally positive in nuclei and distributed as Caspase-3 (Figure 3(f)). They were very rare in proliferating areas and more abundant
in perinecrotic palisades, which were not counted in this work. The percentages of positive cell/nuclei were
constantly <0.02.


### 3.8. Beclin 1 Immunohistochemistry (Performed Only in GBMs)

Beclin 1 was positive in two cases
only. The staining was both nuclear and cytoplasmic (Figure3(d)).


### 3.9. Correlation Analysis

pStat3
and pAkt LIs correlated with the three histological grades (Table3), but not
with survival or with Ki-67/MIB.1 LI within the glioblastoma category. A
correlation of pStat3 LI with Ki-67/MIB.1 LI was not found either comparing the
mean LI values or the frequency peaks. Also pAkt LI did not correlate with
Ki-67/MIB.1 LI. There was a correlation between EGFR amplification status and
EGFRwt and EGFRvIII immunohistochemistry (P  =  .0239 and P  =  .0403, resp.), considering scores B  +  C, but not between EGFRwt
and EGFRvIII immunohistochemistry (Table 2). EGFR amplification and
immunohistochemistry did not correlate with pStat3. In contrast, correlations
were found between EGFRwt immunostaining and levels of pAkt by Western blotting
(P  =  .010), between the pAkt levels by Western blotting and
immunohistochemistry (r  =  0.53, P  =  .0348), and the pAkt levels by
Western blotting and pStat3 LI by immunohistochemistry (r  =  0.473, P  =  .0146)
(Table 4). The number of cases with positive Beclin 1 was too few, that is,
the opposite with the number of cases positive for pStat3, pAkt and Ki-67/MIB.1
LIs in glioblastomas.Table 3
Immunohistochemical frequencies in the
three glioma grades.
Activated pathwayAstrocytoma (n=20)GBM (n=34)Oligodendroglioma (n=10)Grade IIGrade IIIGrade IVGrade IIGrade IIIStat30%50%56.2%0%20%Akt0%20%80%0%20%Table 4
Correlation of EGFR amplification,
EGFRwt and EGFRvIII immunopositivity with pAkt
and pStat3 immunohistochemistry.
Activated pathwayEGFR amplificationP valuePositive (n=12)Negative (n=22)Akt IHC3 (25.0%)8 (36.4%)nsAkt WB*5 (41.7%)9 (41%)nsStat36 (50.0%)8 (31.8%)nsEGFRwt (B  +  C) ImmunohistochemistryPositive (n=22)Negative (n=12)Akt IHC7 (31.8%)4 (33.3%)nsAkt WB*12 (67%)2 (12%).010Stat39 (40.90%)5 (41.6%)nsEGFRvIII (B  +  C) ImmunohistochemistryPositive (n=9)Negative (n=25)Akt IHC3 (33.3%)8 (32.0%)nsAkt WB*3 (33%)11 (44%)nsStat35 (55.5%)9 (36.0%)nsns = not significative.*Only WB values >0.4 were considered.The
values of Caspase-3 and cleaved-PARP1 LIs were so low as to make the correlation study with the antigens
studied be ineffective.In
glioblastomas, no correlation between pStat3, pAkt, Ki-67/MIB.1 LIs and
survival was found by Kaplan-Meier analysis.


## 3.1. EGFR Amplification and Immunohistochemistry (Performed Only in GBMs)

Twelve out of 34 GBMs showed EGFR amplification (35.3%), and 22 of the
GBMs were immunopositive for EGFRwt (64.7%). All but one of the 12 cases that
carried EGFR amplification were immunopositive for EGFRwt
(P  =  .239). Out
of the 22 cases that did not have EGFR amplification, 11 were immunopositive
for EGFRwt (score B  +  C).Out of the 12 cases with EGFR amplification, six were positive for
EGFRvIII (50%) (P  =  .0403), whereas out of the 22 cases without
amplification, only three were positive for EGFRvIII (13.6%) (Table 2).
Immunostaining for EGFRwt was either moderate or intense in most of the cells
analyzed (Figure 1(a)). Immunostaining for EGFRvIII was moderate, and showed
greater regional variability than the EGFRwt immunostaining (Figure 1(b)).Table 2
Correlation of EGFR gene
amplification status with EGFRwt and EGFRvIII immunohistochemistry in 34
glioblastomas.
EGFR amplificationEGFRwt (B  +  C) immunohistochemistryP valueEGFRvIII (B  +  C) immunohistochemistryP valuePositive (n=22)Negative (n=12)Positive (n=9)Negative (n=25)Positive12 (35.3%)11 (91.7%)1 (8.3%).02396 (50.0%)6
(50.0%).0403Negative22 (64.7%)11 (50.0%)11 (50.0%)3 (13.6%)19 (86.4%)(a)
Positive cells for EGFRwt, GBM. (b) Positive cells for EGFRvIII, GBM. (c) PTEN
positive staining, GBM. (d) Ki-67/MIB.1 high number of positive nuclei, GBM.
(e) pStat3 positive nuclei in GBM. (f) pStat3 negative nuclei in grade II
oligodendroglioma. DAB,400×.
(a)(b)(c)(d)(e)(f)Our cases of
glioblastoma were at their first surgery and none derived from a previous
astrocytoma. However, a distinction between primary and secondary glioblastoma
was not made, also because the only EGFR amplification is not an absolute
criterium.


## 3.2. PTEN Mutation Analysis and Immunohistochemistry (Performed Only in GBMs)

All the coding region and the intron-exon boundaries of the PTEN gene were amplified in
ten different PCR fragments in the 34 GBM cases.Direct sequencing led to the
identification of four different single nucleotide variations, located in the
coding region (n=1) and introns (n=3), respectively. These variations were
observed in the four patients resulted negative for PTEN expression by
immunohistochemistry.Variation in the coding region is a non-synonymous
substitution c.389G→A leading to an amino acid substitution at codon 130
(Arg130Gln), previously confirmed as somatic variant in high-grade gliomas and
in gliosarcomas [19, 20]. Among the three intronic variations, one is a new
sequence variation, namely IVS8+36C/T; the remaining were IVS8+32T/G and
IVS1−97A/G, corresponding to validated SNPs in public database of single nucleotide
polymorphisms (http://www.ncbi.nlm.nih.gov/SNP/) (rs555895 and rs1903858,
resp.) (Figure 2). The position of the intronic variations is relative
to the first (+1) or the last (−1) nucleotide of each intron (Gene Bank
sequence NM_000314).Nucleotide sequence analysis of PTEN exon 5. (a) The nonsynonymous substitution (Arg130Gln, CGA  >  CAA) from the tumor DNA of Case
6463. (b) The wild-type exon 5 sequence from normal control DNA.
(a)(b)Thirty out of the 34 GBMs were immunopositive for PTEN (88.2%). The
staining intensity was strong and uniform in all of the cases. Reactive
astrocytes were immunopositive for PTEN, and this staining was also seen in the
four tumors that were negative for PTEN immunostaining (Figure1(c)).


## 3.3. Ki-67/MIB.1

The LI was 5% (2–6%) for grade II astrocytomas, 12% (5–20%) for grade
III astrocytomas, 2% (0–10%) for grade II
oligodendrogliomas, 15% (12–28%) for grade
III oligodendrogliomas, and 23% (12–30%) for GBMs
(Figure1(d)).


## 3.4. Phospho-Stat3 Immunohistochemistry

pStat3 immunostaining was scored as “A” in almost all of the grade II
astrocytomas and oligodendrogliomas, as “B” in the grade III astrocytomas and
oligodendrogliomas, and as “B” or “C” in GBMs. The pStat3 staining was mainly
nuclear, with the exception of few scattered areas where it was cytoplasmic
(Figure1(e)). Cases with positivity <20%
were considered as negative. The percentages of positive cells were 0%, 50%,
and 56.2% for grade II, III astrocytomas and GBMs, respectively. In
oligodendrogliomas, the values were 0% and 20% for grade II and III,
respectively (Figure 1(f)).


## 3.5. Phospho-Akt Immunohistochemistry

In grade II astrocytomas and oligodendrogliomas, the staining was mostly
nuclear and was scored as “A”. In grade III astrocytomas and
oligodendrogliomas, the staining was still nuclear and was scored as “A”. In
GBMs, the staining was mainly cytoplasmic, and only occasionally nuclear, and
was scored as “C” (Figure3(a)). In GBMs
microvascular proliferations were negative and occasionally the staining was
more intense in cells around vessels and outside pseudopalisades. The frequency
values were 0%, 20%, and 80% for grade II, III astrocytomas and GBMs,
respectively. In oligodendrogliomas, the values were 0% and 20% for grade II
and III, respectively (Figure 3(b)).(a) Positive cytoplasmic staining for pAkt, GBM. (b)
Positive nuclear staining for pAkt in grade III oligodendroglioma. (c)
Caspase-3 positive nucleus, GBM. (d) Beclin 1 positive cytoplasmic and nuclear
staining, GBM. (e) PARP1 positive nuclei, GBM. (f) Cleaved-PARP1 positive
nucleus, GBM. DAB,400×.
(a)(b)(c)(d)(e)(f)


## 3.6. Phospho-Akt Western Blotting (Performed Only in GBMs)

The Western blotting analysis showed
variably positive bands (Figures4(a)-4(b)).(a) Western blotting of pAkt expression. Positive
bands in a sample of 8 cases. (b) Quantitative analysis of pAkt levels normalized toβ-actin data.
(a)(b)


## 3.7. Caspase-3, PARP1, and Cleaved-PARP1 Immunohistochemistry (Performed Only in GBMs)

Caspase-3 was positive as cytoplasmic
or nuclear staining or in apoptotic bodies (Figure3(c)). PARP1 was positive in all
the nuclei of the tumor (Figure 3(e)). Cleaved-PARP1 was
occasionally positive in nuclei and distributed as Caspase-3 (Figure 3(f)). They were very rare in proliferating areas and more abundant
in perinecrotic palisades, which were not counted in this work. The percentages of positive cell/nuclei were
constantly <0.02.


## 3.8. Beclin 1 Immunohistochemistry (Performed Only in GBMs)

Beclin 1 was positive in two cases
only. The staining was both nuclear and cytoplasmic (Figure3(d)).


## 3.9. Correlation Analysis

pStat3
and pAkt LIs correlated with the three histological grades (Table3), but not
with survival or with Ki-67/MIB.1 LI within the glioblastoma category. A
correlation of pStat3 LI with Ki-67/MIB.1 LI was not found either comparing the
mean LI values or the frequency peaks. Also pAkt LI did not correlate with
Ki-67/MIB.1 LI. There was a correlation between EGFR amplification status and
EGFRwt and EGFRvIII immunohistochemistry (P  =  .0239 and P  =  .0403, resp.), considering scores B  +  C, but not between EGFRwt
and EGFRvIII immunohistochemistry (Table 2). EGFR amplification and
immunohistochemistry did not correlate with pStat3. In contrast, correlations
were found between EGFRwt immunostaining and levels of pAkt by Western blotting
(P  =  .010), between the pAkt levels by Western blotting and
immunohistochemistry (r  =  0.53, P  =  .0348), and the pAkt levels by
Western blotting and pStat3 LI by immunohistochemistry (r  =  0.473, P  =  .0146)
(Table 4). The number of cases with positive Beclin 1 was too few, that is,
the opposite with the number of cases positive for pStat3, pAkt and Ki-67/MIB.1
LIs in glioblastomas.Table 3
Immunohistochemical frequencies in the
three glioma grades.
Activated pathwayAstrocytoma (n=20)GBM (n=34)Oligodendroglioma (n=10)Grade IIGrade IIIGrade IVGrade IIGrade IIIStat30%50%56.2%0%20%Akt0%20%80%0%20%Table 4
Correlation of EGFR amplification,
EGFRwt and EGFRvIII immunopositivity with pAkt
and pStat3 immunohistochemistry.
Activated pathwayEGFR amplificationP valuePositive (n=12)Negative (n=22)Akt IHC3 (25.0%)8 (36.4%)nsAkt WB*5 (41.7%)9 (41%)nsStat36 (50.0%)8 (31.8%)nsEGFRwt (B  +  C) ImmunohistochemistryPositive (n=22)Negative (n=12)Akt IHC7 (31.8%)4 (33.3%)nsAkt WB*12 (67%)2 (12%).010Stat39 (40.90%)5 (41.6%)nsEGFRvIII (B  +  C) ImmunohistochemistryPositive (n=9)Negative (n=25)Akt IHC3 (33.3%)8 (32.0%)nsAkt WB*3 (33%)11 (44%)nsStat35 (55.5%)9 (36.0%)nsns = not significative.*Only WB values >0.4 were considered.The
values of Caspase-3 and cleaved-PARP1 LIs were so low as to make the correlation study with the antigens
studied be ineffective.In
glioblastomas, no correlation between pStat3, pAkt, Ki-67/MIB.1 LIs and
survival was found by Kaplan-Meier analysis.


## 4. Discussion

Demonstration of pStat3 expression in gliomas using immunohistochemistry
is not an easy task, especially when attempting to quantitatively evaluate it,
due to its heterogeneous expression, which is particularly evident in malignant
gliomas. It has been emphasized that this is particularly important in
microarray-based studies, since it is crucial that the foci examined are
representative of the entire tumor [10]. Heterogeneity in expression can lead to bias when evaluating many antigens in malignant
gliomas. There have been attempts to overcome
this limitation by the introduction of sophisticated procedures to evaluate
positive cells or nuclei when performing immunohistochemical analysis. We used a double-check procedure, and examined
antigen expression in full-size tissue sections and in foci with the highest LI
values.In systemic tumors, it has been debated whether pStat3
levels correlate with a worse or a better prognosis, as it has been observed in
breast cancer [21]. In our series, pStat3 expression is of moderate intensity,
has a nuclear location and its LI significantly increases with malignancy. In
glioblastomas, the LI reaches 56.2%, which is less than the values observed in
previous studies [7], but is similar to the
values that have been reported in more recent studies [11]. As pStat3 is involved in the transformation process,
it should have a predictive role for the whole group of astrocytic gliomas, and
our results confirm that it does have prognostic significance when comparing
the three grades of malignancy. However, pStat3 was not shown to be predictive
within the group of malignant gliomas, as it was observed in a previous study
[11]. No correlation was found between
pStat3 and Ki-67/MIB.1 LI in glioblastomas. The lack of a predictive role for
pStat3 in glioblastomas is consistent with observations that many other
phenotypic features do not have predictive value. The prognostic role of Ki-67/MIB.1 LI itself in glioblastomas has also been the subject of discussion, and we believe that it is not predictive [22]. Our results
are not entirely surprising, since the
relationship between pStat3 and proliferation is rather indirect, and is
mediated through the inhibition of apoptosis by the activation of the Bcl-2
family [23].pStat3 inhibition has been shown to have a number of
anticancer effects. pStat3 upregulates the transcription of several genes that
control tumor cell survival, resistance to apoptosis, cell cycle progression
and angiogenesis [3, 5, 6, 23–25], and pStat3 inhibition specifically
induces apoptosis [3, 4, 8, 26].We found a correlation between EGFR amplification and EGFRwt and
EGFRvIII immunostaining in our samples. Only one case with an EGFR
amplification was negative for EGFR immunohistochemistry, whereas 50% of the
cases without EGFR amplification were positive for EGFR immunohistochemistry,
and 50% of the cases with EGFR amplification expressed EGFRvIII, in contrast to
13.6% of the cases without amplification. Some of these results are consistent
with previous observations [11]. An early hypothesis suggested that pStat3 is activated by deregulation
of EGFR and aberrant IL-6 expression [4]. In gliomas, it was observed that the
levels of pStat3, together with pAkt, correlate with EGFR aberrations, but
specifically with EGFRvIII expression [11]. We did not find any correlation between pStat3 LI and EGFR status. We can only point out that the P value of EGFR amplification when compared with pStat3 LI was the lowest of the
other nonsignificant comparisons. However, it cannot be ruled out that the
small number of samples in our study may have
prevented us from finding a potential correlation between pStat3 and EGFR
amplification, particularly as pAkt levels correlate with EGFR in a Western blotting
analysis. This can be worth also for the
lack of correlation between EGFR status and pStat3 by immunohistochemistry. It
is to be considered that only four cases of GBMs showed negative PTEN expression,
and only one had a real mutation. Anyway, all the four cases without PTEN
expression showed already EGFR amplification, with the exception of the only
case with a real PTEN mutation.The impossibility to find any correlation of pStat3
expression with the apoptotic index was due to the short duration of this event
and to the too low number of apoptotic nuclei found in proliferating tumor
areas, which can be related to the different pathways leading to apoptosis. The action of pStat3, via the Bcl-2 family [5, 23], may lead to the
inhibition of transcriptional or intrinsic apoptosis pathway, and may not
affect receptor-mediated or extrinsic apoptosis pathway, which may be the major
one in perinecrotic tumor areas [27]. The selection of only proliferating areas in our study, and the
exclusion of regressive and necrotic ones, may have eliminated the major
sources of apoptosis. There is
cross-talk between the intrinsic and extrinsic pathway to apoptosis by BH3-interacting
domain death agonist (BID) [28]. However, the low detectable values of
Caspase-8 in the receptorial pathway to apoptosis in gliomas [29], would make
pointless this cross-talk.Even
considering that PTEN was mutated only in one case of our series, no
correlation was found between pAkt LI and Ki-67/MIB.1 LI. The levels of pAkt
correlate with malignancy within the three grades in our experiments and in other reports [30], but in glioblastomas there is no correlation with the
proliferation marker. The explanation can be the same given before for pStat3.The
localization of pStat3 is not so clear-cut in the various observations. It was
also shown to be predominantly localized in endothelial cells [31], or in tumor
and in endothelial cells [7]. Activated Stat3alpha found in brain tumors was thought
to be the result from the action of the endothelial tyrosine kinase VEGFR-2,
which plays a central role in autocrine VEGF activation [31]. We found
occasional endothelial cells in vascular proliferations of GBMs
that stained positive for pStat3, but we could not correlate them with other
features of the tumors.The
correlation of pStat3 with pAkt found by us confirms previous observations
[11], and emphasizes the position of pStat3 downstream pAkt in the PI3K/Akt
signalling pathway [32]. Finally, Beclin 1 was found to be positive in two
cases only of glioblastomas, with an inverse correlation with pAkt and pStat3.
Beclin 1 is considered to be representative of the type II programmed cell
death, or autophagy, and in cancers this pathway is not clearly separated from
type I programmed cell death, or apoptosis, and both pathways can be functional in
the same cell [33]. In many malignancies, Beclin 1 is monoallelically deleted
and is under-expressed. In brain tumors, Beclin 1 levels have been
found to decrease with malignancy. In glioblastomas its expression is reduced
in comparison with low-grade gliomas, and is rather nuclear indicating a loss
of gene function [34]. Our almost negative findings in glioblastomas are
consistent with these observations and also with the high expression of pStat3
found in the same cases. In this regard, the induction of autophagy by
inhibitors of mTOR [35] and pStat3 [14], in addition to radiation and
chemotherapy with temozolomide [36–38], may be significant for therapeutic approaches.



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