Nucleolar Organizer Regions in Squamous Cell
Carcinomas of the Uterine Cervix
Treated with Chemoradiotherapy
a, bIin Kurnia, aYoshiyuki Suzuki, cBudiningsih Siregar, dAndri Andrijono,eIrwan Ramli, eCholid Badri, aYukari Yoshida, aTakashi Nakano.
aDepartment of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan. bDepartment of Biomedical, Center for Technology of Radiation Safety and Metrology, National Nuclear Energy Agency, Jakarta, Indonesia. cDepartment of Pathology Anatomy, Faculty of Medicine, University of Indonesia/Cipto Mangun Kusumo Hospital, Jakarta, Indonesia. dDepartment of Obstetric and Gynaecology, Faculty of Medicine, University of Indonesia/Cipto Mangun Kusumo Hospital, Jakarta, Indonesia. eDepartment of Radiotherapy, Faculty of Medicine, University of Indonesia/Cipto Mangun Kusumo Hospital, Jakarta, Indonesia.
ABSTRACT
Background: Nucleolar organizer regions (NORs) are chromosomal loops of DNA involved in ribosomal synthesis. Their size and number in a nucleus has been reported to reflect the proliferative activities of various cells. The purpose of this study was to assess the meaning of the number of NORs in tumor-cell nucleoli compared with other proliferative markers before and after 10 Gy irradiation.
Materials and Methods: Forty six biopsy specimens from twenty three patients with cervical squamous cell carcinoma treated with chemoradiotherapy at Cipto Mangun Kusumo
Keywords:AgNOR, cervical squamous cell carcinoma, chemoradiotherapy, MIB-1.
Hospital (Indonesia) taken before radiotherapy and after exposure to 10 Gy were analyzed. The number of NORs was measured using a silver staining method. MIB-1 and p53 labeling indexes (LI) were measured using an immunohistochemical method. Histological radiation response and mitotic index (MI) were investigated with hematoxylin and eosin staining.
Result: After 10 Gy irradiation, the number of NORs per nucleolus (AgNOR score) decreased from 4.9 to 3.4 (p<0.001). MIB-1-LI and MI increased from 26 to 39 (p<0.01) and from 0.006 to 0.012 (p<0.001), respectively. Before radiation therapy, a positive correlation was found between AgNOR score and MIB-1-LI (p=0.001) and between AgNOR score and MI (p=0.01). At 10 Gy, there was no significant correlation among number of NORs, MIB-1-LI, MI, or p53-LI. Histological radiation response had no correlation with AgNOR, MIB-1-LI, MI, or p53-LI.
Nucleolar organizer regions (NORs) are chromosomal loops of DNA involved in ribosomal synthesis.1 The silver staining technique can easily detect NORs in formalin-fixed, paraffin embedded tissues and NORs can be identified as black dots in the nucleolus (AgNORs).2 This method permits the rapid evaluation of morphology and tumor cell kinetics even using small biopsies. Evaluation of AgNORs parameters (number, size, and distribution) has been applied in tumor pathology both for diagnostic and prognostic purposes.3 Their size and number have been reported to reflect the proliferative activity of tumor cells.4 The quantity and distribution of NORs have been used for the differential diagnosis of malignant versus benign cells5-8 and for cancer prognosis.9-14
The malignant grades of tumor cell and proliferative activity have been assessed using immunohistochemical staining with several cell cycle related parameters.15-18 MIB-1, also known as Ki-67, is expressed in all cell cycle stages except for G0 and early G1 phase. This antigen is thought to be associated with a nuclear antigen protein-DNA replicase complex, similar to protein-DNA topoisomerase II.19 Generally, a higher MIB-1 labeling index (LI) correlates with worse prognosis; however, tumors with higher MIB-1-LI are often radiosensitive. Tumors with higher MIB-1-MIB-1-LI before radiotherapy showed better response than those with lower MIB-1-LI, and also higher MIB-1-LI index at 9 Gy could be used as a favorable prognostic factor in patients with cervical squamous cell carcinoma treated by radiotherapy.20 Hence, the meaning of MIB-1-LI in radiation therapy is still equivocal.
Mitotic Index (MI) is also a well known parameter to estimate cellular proliferation, which counts the number of cells in the mitotic phase and has been widely applied as part of some tumor grading schema.21 Higher mitotic index is associated with shortened disease free survival in breast cancer22 and with adverse events (recurrence, metastasis, or tumor-related death) in uterine smooth muscle tumors.23 MI also shows an inverse correlation with local control and with the prognosis of cervical cancer treated with radiotherapy.16
The p53 gene, present on chromosome 17p, acts as a tumor-suppressor gene, controlling entry into the S-phase of the cell cycle. The p53 gene plays important roles in cell proliferation. Mutation of this gene inactivates its suppressor activity and is related to tumor progression.24 High p53 labeling index (p53-LI) before radiotherapy was correlated with the poor survival of cervical cancer patients after radiotherapy.25 Another report found no relationship
immunohistochemically detected p53 protein in cervical cancer before treatment.26, 27 However, the correlation between the number of NORs and other proliferative markers (MIB-1-LI, MI, and p53-LI) remains debatable. Therefore, the current study was performed to assess the meaning of the number of NORs using a simple silver staining technique method, comparing with MIB-1-LI, MI, and p53-LI before and after 10 Gy irradiation.
MATERIALS AND METHODS
Patients’ characteristics
A total of 46 biopsy specimens taken before chemoradiotherapy and at one week after the initiation of radiotherapy (which corresponded to 10 Gy) were investigated from 23 patients with squamous cell carcinoma of the cervix. The patients were treated with chemoradiotherapy at Cipto Mangunkusumo Hospital (Jakarta, Indonesia) from 2005 to 2006. Their ages ranged from 39-65 years old (mean and median: 49±7 and 48 years old, respectively) with clinical stage from 1b to 4a (Table 1).
Chemoradiotherapy protocol
Patients were treated with a combination of external beam radiotherapy (EBRT) with 60Co gamma rays and 192Ir high-dose-rate intracavitary brachytherapy (HDR-ICBT). EBRT was administered to the whole pelvis with a clinical target volume that included the primary cancer, uterus, internaliliac, presacral, upper external iliac, and lower common iliaclymph nodes. This was usually achieved by a “four-field box technique,” or sometimes by anteroposterior and posteroanterior parallel opposed portals. The usualfield borders for anterior and posterior fields were superiorat the L4-L5 interspace, inferior at the bottom of the obturator foramen or 2 cm distal from the tumor, and 1.5 to 2.0 cm lateral to the bony pelvicwall. Lateral fields had the anterior border at the symphysispubis and the posterior border encompassed the entire sacral silhouette. A total dose of 50 Gy was prescribed in 25 equal fractions to the isocenter. HDR-ICBT using a Microselectron (Nucletron International, Amsterdam, Netherlands) followed EBRT in two fractions (850 cGy/fraction) at point A.28, 29, 30
T
able1. P
atients characteristics of this research.
N o Clinical A ge A
gNOR BT
MIB-1-LI BT M IB T p53-LI BT AgNOR A T M IB -1 -L I A T M I A T p53-LI A T Shimosato stage
Oboshi at A
T 1 4a 42 5. 0 31 0.007 25 3. 2 37 0.012 33 0 2 2a 45 4. 7 18 0.003 7 3. 6 30 0.005 1 1 3 3b 52 5. 5 35 0.007 19 4. 3 41 0.030 16 1 4 3b 43 4. 8 22 0.005 8 3. 6 36 0.011 8 2A 5 3a 45 3. 6 24 0.010 0 3. 2 39 0.013 0 1 6 1b 57 5. 3 34 0.004 8 4. 0 43 0.009 5 0 7 3b 65 4. 9 16 0.004 16 4. 1 38 0.013 14 1 8 3b 42 5. 1 17 0.002 13 2. 3 32 0.010 0 2B 9 2b 45 5. 4 26 0.006 6 3. 7 39 0.008 0 2B 10 3b 39 5. 1 23 0.006 0 3. 2 55 0.013 0 2B 11 2b 50 4. 4 27 0.006 7 2. 8 41 0.014 12 2B 12 3b 58 3. 6 16 0.004 2 2. 3 38 0.010 0 1 13 2a 57 5. 1 18 0.005 15 2. 1 40 0.017 17 2A 14 2b 50 4. 5 29 0.005 6 2. 9 40 0.013 2 2A 15 3b 48 3. 3 17 0.004 7 2. 9 34 0.006 3 2A 16 3b 55 5. 4 31 0.009 16 4. 1 40 0.014 0 0 17 3b 45 7. 3 33 0.011 1 5. 2 35 0.010 0 2A 18 1b 46 4. 2 20 0.004 4 3. 3 39 0.009 21 0 19 2b 46 6. 8 36 0.013 0 3. 5 45 0.007 6 0 20 2b 50 4. 7 26 0.003 15 3. 5 40 0.015 1 2B 21 3b 48 3. 1 15 0.003 1 2. 8 31 0.008 14 1 22 2b 52 5. 2 33 0.004 10 4. 2 35 0.010 12 0 23 2b 40 6. 5 41 0.012 0 3. 4 51 0.024 1 1 B T
: before treatment, A
T
exceeded1.5×109/L, and the platelet count exceeded 100×109/L. Patientsreceived transfusions where necessary to maintain a hemoglobin levelgreater than 110 g/L.28, 29, 30
Histopathological Study
Simple silver staining technique and AgNOR
score:
Nucleolar organizer regions (NORs) are chromosomal segments, which contain ribosomal genes. NORs also contain a set of acidic, non-histone proteins that bind silver ions and are selectively visualized by silver stain methods in routinely processed cyto-histological samples.31 The NORs can be identified as black dots in the nucleolar area and are called AgNORs.3 A simple silver staining technique was performed in accordance with the technique described by Ploton et al.33 Tissue sections were cut at 4μm thick from formalin fixed, paraffin-wax embedded blocks. The sections were dewaxed in xylene and hydrated through decreasing grades of ethanol followed by through washing in deionized water for 8–10 minutes. The staining solution was prepared by dissolution of powdered gelatin at a concentration of 2% w/v in deionized water over waterbath at 60–700C. Pure formic acid was then added to a final concentration of 1%. This solution was mixed 1:2 (v/v) with 50% aqueous silver nitrate solution (freshly made each time), filtered through a 0.22 μm Millipore filter, and dropped onto the slide-mounted section. The sections were incubated in the dark for 40–45 minutes at room temperature. After rinsing three times with deionized water, the slides were immersed for 10 minutes in 5% sodium thiosulphate solution, dehydrated in ascending ethanol concentrations, cleared with xylene, and mounted.
According to the recommendations of Crocker et al.,32 dots lying in a group or cluster (almost aggregated or partly disaggregated) were treated as one structure, where AgNORs could been seen separately they were considered as individual AgNOR.
MIB-1 and p53 immunohistochemical
staining:
MIB-1 and p53 staining was performed using an immunohistochemical technique. Tissue sections were cut at a thickness 4 μm, dewaxed in xylene, and hydrated through grades of ethanol followed by PBS (5 minutes each). The sections were incubated in DAKO Target Retrieval Buffer (pH 6: DakoCytomation, Denmark) in a microwave oven at 940C for 20 minutes, cooled to room temperature for 20 minutes, and washed in PBS (3 times for 5 minutes
Peroxidase Block (DakoCytomation) for 10 minutes, followed by PBS for 10 minutes, and incubation with MIB-1 (DakoCytomation) and anti-p53 antibodies (DakoCytomation) overnight at 40C. After incubation with MIB-1 and p53 antibodies, the sections were washed with PBS for 15 minutes, and incubated with secondary antibody (Labeled Polymer HRP (DakoCytomation)) for 60 minutes at room temperature. Then, the sections were washed with PBS for 10 minutes, developed with 3,3'-diaminobenzidine tetrahydrochloride for 30 seconds, slightly counterstained with hematoxylin, dehydrated, and mounted.
Hematoxylin-eosin staining for assessing MI
and histological response
An experienced senior pathologist reviewed the histopathological slides. The histopathological grading was done on formalin-fixed and paraffin-embedded specimens, which were cut into 4 μm-thick serial sections, deparaffinized, and dehydrated. The sections were stained with hematoxylin and eosin to examine MI and the histopathological radiation effect.
Assessment of AgNOR, MIB-1-LI, MI, and
p53-LI:
AgNOR score (the number of NORs in a tumor cell nucleoli) were counting in 100 cell nuclei using a light microscope (magnification ×400) with the immersion oil method of Crocker et al.32 MIB-1-LI, p53-LI, and MI are the percentages of MIB-1- and p53- positive tumor cells and mitosis, respectively. The sections were evaluated using a blind protocol to prevent biased results. Three randomly selected representative fields were used to count tumor cells to determine the index, a minimum of 1000 tumor cells in every case were counted in three color photographs (original magnification × 400) to minimize variations originating from within sections. One of the authors (IK) blindly performed cell counting. Reproducibility was determined by counting the same color photographs twice in five cases and directly using a light microscope, and variation between the counts was less than 5%, indicating that no bias was introduced.
Assessment of histological radiation effect:
Table 2. Histological grades for radiation effect by Shimosato Oboshi Grade Histologic findings
0 No effect on cancer cell.
I Charactristics are noted in tumor cells, but tumor structures are not destroyed. There are no defects in tumor nests as a result of the dissaperance of tumor cells.
II In addition to characteristic cellular changes, tumor structures have been destroyed as a result of the dissaperance of tumor cells. However, a variable number of viable cells remain.
A. Destruction of tumor cells is mild, viable tumor cells are observed frequently. B. Destruction of tumor cells structures is severe: few viable tumor cells are seen.
III Significantly altered, presumaby nonviable tumor cells are present singly or in small clusters, and few viable cells are seen.
IV No tumor cells remain in any section (local cure).
This assessment was conducted by co author without any clinical date (BS, pathologist).
Statistical Analysis
Student’s t-test was used to determine the difference among AgNOR score, MIB-1-LI, MI, and p53-LI before and after 10 Gy irradiation and to determine the difference AgNOR score, MIB-1-LI, MI and p53-LI in terms of radiation response. The linear fitting was used to determine the regression line of the correlation between of AgNOR score and MIB-1-LI, MI, or p53-LI before and after 10 Gy irradiation.
RESULTS
A. Before chemoradiotherapy
AgNOR score, MIB-1-LI, MI, and p53-LI
AgNOR score varied from 3.1 to 7.3 (mean 4.9±1.0)
(Figure 1a). Tumor cells with a positive reaction to MIB-1 antibody resulted in intense brown staining of the nuclei (Figure 1b). MIB-1-LI varied from 15% to 41% (mean 25±7%). MI varied from 0.002 to 0.021 (mean 0.006±0.003). p53-LI varied from 0% to 25% (mean 8±7%). Figure 2a and 2b shows a positive correlation between AgNOR score and MIB-1-LI (correlation coefficient=0.70, p<0.01), and between AgNOR score and MI (correlation coefficient=0.62, p<0.01) and there was no statistical significant correlation between AgNOR score and p53-LI (correlation coefficient=0.02, p=0.94: figure 2c).
B. After 10 Gy irradiation
AgNOR score, MIB-1-LI, MI, and p53-LI
AgNOR score varied from 2.1 to 5.2 (mean 3.4±0.7). MIB-LI varied from 30% to 55% (mean 39±6%). MI varied from 0.005 to 0.030 (mean 0.012±0.006). p53-LI varied Figure 1: (a) Squamous cell carcinoma of the uterine cervix.
Tumor cells show AgNOR dots in the tumor cell nuclei.
from 0% to 33% (mean 7±8%). After 10 Gy irradiation, AgNOR score decreased (p<0.01), MIB-1-LI increased (p<0.01), and MI increased (p<0.01). However, p53-LI did not change significantly (p=0.58). (Figure 4a-d, respectively). No statistically significant correlation was
Figure 2 (b), and no significant correlation between AgNOR and p53-LI
Correlation between AgNOR Score and Mitotic Index before treatment
r=0.6 p<0.01 y=0.002x-0.003
Figure 2 (c) in squamous cell carcinoma of the uterine cervix before treatment.
Correlation between AgNOR Score and p53-LI before treatment
r=0.02 p=0.94 y=0.1x+7.5
Figure 3. Decreased AgNOR number and increased AgNOR volume after 10 Gy irradiation in cervical cancer treated with chemoradiotherapy. Original Magnification x 400.
Figure 2. Positive correlation was found between AgNORs and MIB-1-LI (a), AgNORs and Mitotic Index
MIB-1-LI before treatment
r=0.7 p<0.01 y=5.3x-0.6
p<0.01 BT = Before treatment
AT = After 10 Gy
BT AT
Figure 4. Decrease in AgNOR (a) increase in MIB-1 index
p<0.01 BT = Before treatment
AT = After 10 Gy
BT AT
Figure 4 (c), no significant change in p53-LI BT = Before treatment AT = After 10 Gy
p<0.01
BT AT
BT = Before treatment AT = After 10 Gy
p=0.58
BT AT
Figure 4 (d) before and after 10 Gy irradiation in squamous cell carcinoma of the uterine cervix treated with chemoradiotherapy.
Table 3. Mean value of AgNOR, MIB-1-LI, MI, p53-LI in squamous cell carcinoma of the uterine cervix before treatment and after 10 Gy irradiation
Before After 10 Gy p value treatment irradiation
AgNOR 4.9±1.0 3.4±0.7 < 0.01
(range) (3.1 - 7.3) (2.1- 5.2)
MIB-1-LI 25±7% 39±6% < 0.01
(range) (15% - 41%) (30 - 55%)
MI 0.006±0.003 0.012±0.006 < 0.01
(range) (0.002 - 0.021) (0.005 - 0.03)
p53-LI 8±7% 7±9% 0.58
(range) (0% - 25%) (0% - 33%)
LI: labeling index, MI: Mitotic index
observed between AgNOR score with MIB-1-LI, MI and p53-LI (p=0.88, 0.67 and 0.74, respectively).
AgNOR score, MIB-1-LI, MI, and p53-LI
with histological radiation effect.
Histological radiation effects were grouped into a poor response group (13 patients: 57%) and a good response group (10 patients: 43%). AgNOR score varied from 2.3 to 4.3 (mean 3.5±0.6) in the poor response group and from 2.1 to 5.2 (mean 3.2±0.9) in the good response group. The MIB-1-LI varied from 30% to 51% (mean 39±5%) in the poor response group and from 32% to 55% (mean 39±6%) in the good response group. The MI varied from 0.005 to 0.030 (mean 0.013±0.007%) in the poor response group and from 0.006% to 0.017% (mean 0.012±0.003%) in the good response group. The p53-LI varied from 0% to 33 % (mean 9±10%) in the poor response group and from 0% to
Table 4. Mean value of AgNOR, MIB-1 LI, MI and p53-LI by histological radiation response in squamous cell carcinoma of the uterine cervix after 10 Gy irradiation
Histological radiation response
Good Bad p value
response response
AgNOR 3.2 ± 0.9 3.5±0.6 0.35
(range) (2.1 - 5.2) (2.4 - 4.3)
MIB-1-LI 39 ± 6 % 39±5 % 0.93
(range) (32% - 55 %) (30% - 51%)
MI 0.012±0.003 0.013±0.007 0.74
(range) (0.007- 0.017) (0.005 - 0.030)
p53-LI 4 ± 6 % 9 ± 10% 0.15
(range) ( 0% - 17%) (0% - 33%)
LI: labeling index, MI:Mitotic index
17% (mean 4±6%) in the good response group. There were no statistically significant differences between AgNOR score, MIB-1-LI, MI, and p53-LI in terms of the histological radiation response (p=0.35, 0.93, 0.74 and 0.15, respectively) (Table 4).
DISCUSSION
larger in the phases of the cell cycle from S to G2.36 MIB-1 was present in all cell cycle stages except for G0 and early
G1.36, 37 Dong et al.23 reported that the AgNOR score in
human cervical squamous cell carcinoma cell line showed a strong correlation with MIB-1-LI. They also reported that both AgNOR score and MIB-1-LI showed inverse correlations with potential doubling time and the length of S phase. A significant correlation among these different parameters suggests that AgNOR score is an important parameters related to proliferative activity.
The increase in MIB-1-LI after 10 Gy irradiation is regarded as a recruitment phenomenon that means a proportion of G0 cells entered G1 and S phase. The same phenomenon was reported by Nakano et al. in a study on cervical squamous cell carcinoma treated by radiotherapy15 and by Kurnia et al. in a study on cervical squamous cell carcinoma treated by chemoradiotherapy and radiotherapy alone.39 As for NORs, the number and size of NORs are highly variable within the nucleolus according to rRNA transcriptional activity.37 The amount of nucleolin was not substantially modified during the first 9 hours of regeneration, and the amount of nucleolin was 1.5-fold larger than the control at 12 hours after regeneration, and increased progressively reaching a maximum value at 18 to 21 hours after regeneration before the cell enters mitosis.40 Quantitative changes of B23 protein levels were seen from 15 hours after regeneration.36 Ielmini et al.41 reported that the number of NORs decreased after irradiation but the volume increased until 5 days after irradiation in biopsy tissues of rat tail cells. Schwint et al.42 reported that AgNOR score decreased after irradiation while the volume of AgNORs increased. In this study, a decrease in AgNOR score was also found. The rise in single AgNOR volume would indicate transcriptional activation of previously in-active NORs,42 and the reduction in number of AgNOR per nucleus may indicate association processes.43 However, the reduction the number of AgNOR and increase in AgNOR volumes cannot be explained by association processes alone but results mainly from an absolute rise in transcriptional activity.42 These events would lead to an increase in ribosomal RNA and, ultimately, to arise in protein synthesis41. Sirri et al.44 in a study on stimulated rat hepatocytes found that the major AgNOR proteins increase in S-phase and continuously accumulate until the end of the G2 phase. We think this phenomena also related with the process of G2 block of the cell after received irradiation.
The current study could not find any relationship between AgNOR index and MIB-1-LI and p53-LI. Some studies have reported a correlation between p53 immunopositivity and MIB-1,45 but other studies have failed
tendency for higher AgNOR score or MIB-1 in p53 immunopositive compared with p53 immunonegative samples from carcinoma of the extrahepatic bile duct. Pich et al.,49 reported that p53 immunopositivity was associated with high AgNOR quantity and MIB-1-LI in male breast carcinoma and in papillary superficial bladder neoplasm. Karokok et al.,50 found no statistical relationship between AgNOR score and p53-LI in bladder carcinoma.
The present study showed no statistical significant difference of AgNOR score, MIB-1-LI, and MI between good and bad histological radiation response. Nakano et al.16 reported, cervical cancer patients with MIB-1-LI 33% or greater showed significantly better histological radiation response at 30 Gy irradiation than patients with those less than 33%.Begum,51 reported that a remarkable histological change in cervical cancer could be seen at 30 Gy irradiation. We think the difference of radiation effect in histological level between higher 1-LI and lower MIB-1-LI have not been seen at10 Gy irradiation, if there were any difference, it may still exist in the cellular level. We also think that some factors like biopsied specimen, heterogeneity of tumor tissue, sampling errors, and number cases of this study also influenced the statistical significant of AgNOR score, MIB-1-LI, and MI between good and bad histological radiation response.
In conclusion, AgNOR score can be used as a proliferation marker for assessing tumor cell proliferation activity before chemoradiotherapy, but not after 10 Gy irradiation.
ACKNOWLEDGEMENT
This research was supported by a Research Grant from Center for Technology of Radiation Safety and Metrology, National Nuclear Energy Agency in Indonesia (2005-2006) and Japanese Society for Promotion Science (JSPS) in Japan. This research was approved by the Committee for Medical Research Ethics of the Faculty of Medicine University of Indonesia. The authors thank Kunto Wiharto, MD (National Nuclear Energy Agency, Indonesia), Endy Moegni, MD, Endang SR Harjolukito, MD, Prof. Soehartati Gondhowiardjo, MD, PhD, and Prof. Raden Susworo, MD, PhD (University of Indonesia) for scientific advice and to Mr. Heri Basuki, Mrs. Aziz Yusnelly, Mrs. Arena Said (University of Indonesia), Abdul Mobaraki, MD, and Wael Al-Jahdari, MD, PhD (Gunma University) for technical assistance.
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