Prognostic Impact Of pAkt In Glioblastoma Treated With Radiation Therapy

1
Prognostic Impact Of pAkt In Glioblastoma Treated
With Radiation Therapy

• Yoshiyuki Suzuki MD1, Katsuyuki Shirai MD1,
  Kuniyuki Oka MD2, Yukari Yoshida PhD1,
  Shin-ei Noda MD1, Masahiko Okamoto MD1,
• Yoshihiko Suzuki MD3, Jun Itoh MD4,
  Hideaki Itoh MD5, Shogo Ishiuchi
  MD6, Takashi Nakano MD1
• Department of Radiation Oncology, Gunma
  University Graduate School of Medicine, Maebashi,
  Japan. 2) Department of Pathology, Mito
  Saiseikai General Hospital, Mito, Japan.
• 3) Division of Radiology, Takasaki National
  Hospital, Takasaki, Japan 4) Section of
  Radiology, Maebashi Red Cross Hospital, Maebashi,
  Japan
• 5) Section of Pathology, Maebashi Red Cross
  Hospital, Maebashi, Japan. 6) Department of
  Neurosurgery, Gunma University Graduated School
  of Medicine, Maebashi, Japan.
  

Conclusion
Objection
High expression of Phospho-Akt (Ser473) predicts
a poor prognosis in patients with
glioblastoma.
Phospho-Akt (pAkt) plays an important role in
tumorigenesis through promotion of cell survival
and mediating cell proliferation. High
expression of pAkt has been reported to be
associated with an unfavorable prognosis in
prostate cancer, breast cancer, and lung cancer
etc.. However, the prognostic value of pAkt
expression in patients with glioblastoma is still
equivocal. Here, we investigated the prognostic
value of pAkt expression in glioblastomas treated
with radiation therapy by using
immunohistochemical methods.
Results
Glioblastomas showed either or both
cytoplasmic and nuclear positive findings for
pAkt (Figure 1). The number of cytoplasmic and
nuclear positive specimens were 18 (27.7) and 3
(4.7), respectively (two specimens showed both
cytoplasmic and nuclear positive). A total of
29.7 (19/64) of tissue specimens had greater
than 50 pAkt positivity. The median survival
period of the patients with pAkt positive and
negative tumor were 10 and 14 months,
respectively. Two years overall survival rate of
the pAkt positive and negative patients were 0
and 24.4, respectively. Survival rate of the
patients with pAkt positive tumor was
significantly lower than that of the patients
with pAkt negative tumors (p0.004 Figure 2).
The overall survival based on clinical
characteristics and pAkt expression is shown in
Table 1. The 2-year survival rate of patients on
whom had been performed either gross total
removal or subtotal removal was 30.0,
significantly higher than 7.3 of patients whom
had been performed either partial resection or
biopsy (p0.047). Whereas, the 2-year survival
rates of patients aged lt60 and 60lt were 17.1 and
17.2, respectively (p0.31). The 2-year
survival rates for male and female were 12.2 and
26.1, respectively (p0.48). The 2-year
survival rates of patients irradiated with BED lt
72 and 72lt were 16.3 and 19.0, respectively
(p0.59). The 2-year survival rates of patients
without and with chemotherapy were 11.8 and
20.9, respectively (p0.44). Clinical
characteristics were not significant prognostic
factors. Table 2shows the multivariate analysis
for 2-year survival. The extent of surgery had
the strongest and statistically significant
impact on 2-year survival (standard regression
coefficient0.336, p0.01). The pAkt expression
had the second strongest impact (standard
regression coefficient-0.23, p0.06).
Materials Methods
Reuben JS et al. Nature 441,2006
Patients and
specimens Tissue specimens were obtained from all
of the 65 patients with pathologically confirmed
glioblastoma treated with radiation therapy at
the Gunma University Hospital (Maebashi Japan),
Takasaki National Hospital (Takasaki Japan), and
Maebashi Red Cross Hospital (Maebashi Japan)
between 1982 and 2005 (Patients, who were lost to
follow up within six months, were excluded).
Table 1 shows the clinical characteristics of the
65 patients. The median follow-up period was
11.5 months (range from 4 to 62 months).
Fifty-nine patients died due to tumor
progression, 4 patients were alive more than 3
years and 2 patients were lost by follow-up at 12
months. The mean and median of age were 56.2
16.3 and 53 years old, respectively. Thirty-two
patients were aged gt60 years old and 35 were aged
lt60 years old. The numbers of male and female
patients were 39 and 26, respectively. Gross
total resection was performed in 19 patients,
partial resection in 44 patients, and biopsy in 4
patients. The mean and median radiation dose
were 57.3 8.1 Gy and 60 Gy, respectively.
Regarding the radiation therapy, in general,
conventional therapy (2 Gy/fr./day 5 days/week)
was performed. In some patients, hyper- and
hypo-fractionated radiation therapies were
performed. Therefore, we used the biological
effective dose (BED) for analysis, and set the
a/ß ratio as 10.0 Gy. The mean and median BED
were 96.6 13.2 and 100, respectively. In BED,
51 patients were irradiated with BED gt100 and 16
patients were irradiated with BED lt100.
Chemotherapy was performed in 45 patients as an
initial treatment before and/or after
irradiation. Regarding the histological
diagnosis, there were glioblastoma multiforme in
62 patients, giant cell glioblastoma in 3 and
gliosarcoma in 2, based on the WHO Classification
of Tumors of the Nervous System (2000). We also
investigated the pathological features, including
the extent of necrosis, vascularity, cell density
and existence of giant cells. Immunohistochemistry
The tissue specimens were cut into 4 µm sections
on cylan-coated slides, then were deparaffinized
and hydrated. For antigen retrieval, they were
heated by microwave for 90 minutes in Dako Target
Retrieval Solution (Dako, CA, U.S.A.) at 95.0 C
and were cooled for 20 minutes. After washing
with Tris Buffered Saline (TBS), nonspecific
binding was blocked with peroxidase-blocking
solution (Dako) for 15 minutes. The specimens
were incubated overnight at 4.0 C with
Phospho-Akt (Ser473) Antibody antibody (Cell
Signaling Technology, Beverly, MA) diluted with
antibody diluent (Dako) at 1200. Then, they
were washed with TBS. They were incubated with a
labeled-polymer conjugated second antibody
EnVisionkit (Dako) for 45 minutes. After
washing in TBS, the specimens were developed with
3,3'-diaminobenzidine tetrahydrochloride (Dako)
for 7 minutes. Specimens were slightly
counterstained with hematoxylin, and finally
dehydrated and mounted. As a positive control, a
known positive control (cervical cancer) specimen
was handled in parallel. As a negative control,
a specimen was treated without a primary antibody
in the incubation step. Evaluation All
immunohistochemically stained slides were
pathologically assessed by one of the authors
(K.O), without knowledge of the clinical data.
The cell nuclei and cytoplasm of more than 500
tumor cells were evaluated in each specimen.
Positive staining was defined when more than 50
of either nuclear or cytoplasma of the tumor
cells were stained in each specimen (). The
correlation between pAkt expression and overall
survival were evaluated.
Figure 1 ? Figure 2 ?