Aim: To evaluate the premalignant and malignant lesions of the uterine cervix on light microscopy for apoptosis. To calculate and correlate the apoptotic index (AI), mitotic index (MI) and turnover index of the lesions. Materials and methods: A 2-year retrospective (November 2007 to October 2009) and 1-year prospective study (November 2009 to October 2010). A total of 95 cases of premalignant and malignant lesions of cervix were studied. The hematoxylin-eosin stained slides were screened for apoptosis under oil immersion lens. Apoptotic index (AI) was calculated as the number of Apoptotic cells and Apoptotic bodies, expressed as percentage of total no. of tumour cells counted in each case. Mitotic index (MI) was calculated by counting mitosis among 1000 tumour cells. Turnover index (TOI) was obtained by adding MI and AI, i.e., (TOI = MI + AI). After calculating, all these indices were correlated with different grades of the cervical lesions. Results: Premalignant lesions were divided into four subgroups. On statistical analysis, it was found that the difference in the apoptotic indices and turnover indices in all the four subgroups was not statistically significant. However for mitotic indices, significant statistical difference was found in Subgroup II (mild dysplasia vs. moderate dysplasia) with a p value of 0.03 and in subgroup IV (squamous metaplasia vs. moderate/severe dysplasia) with a p value of 0.0005. Within the malignant group, we compared well differentiated with less well differentiated subgroup. Statistical analysis revealed a significant difference in TOI between the two subgroups of malignant tumours (p = 0.04). Statistical comparison between premalignant and malignant group of lesions showed a highly significant difference between the AI, MI and TOI with p value of 0.0001. Conclusion: We conclude that proliferative and apoptotic indices are useful in distinguishing between benign and malignant lesions of the cervix. Proliferative activity of a lesion is a reliable indicator of its malignant potential and together with apoptotic count gives an idea about the net growth of a tumour.
Apoptosis or programmed cell death is the cell’s intrinsic death program that regulates various physiological as well as pathological processes [
The tendency of a cancer cell to undergo apoptosis may have important implications for tumour progression and response to treatment [
It is generally accepted that electron microscopy is the best way to identify apoptotic cells [
There are many studies available in the literature that have evaluated the apoptosis by light microscopy in variety of human tumours, yet there are very few studies available from India, which have studied the apoptosis with light microscopy in premalignant and malignant lesions of uterine cervix. Cervical cancer is the third most common cancer affecting women worldwide, the most common cancer in women in India and several less developed countries [
Human Papillomavirus is considered to be the most important agent in cervical oncogenesis. Presence of cancer associated HPV and persistent detection of high risk HPV such as HPV 16, 18 are the most important risk factors associated with cervical cancer. Other risk factors are early age at first intercourse, multiple sexual partners, increased parity, a male partner with multiple sexual partners, exposure to oral contraceptives and nicotine; ge- nital infections such as Chlamydia, immune status and low socioeconomic background [
The most common symptoms of cervical cancer are copious foul-smelling vaginal discharge, abnormal bleed- ing or inter-menstrual bleeding, post-coital bleeding, postmenopausal bleeding or backache [
The present study is a retrospective study for a period of 2 years from November, 2007 to October, 2009 and prospective study from November 2009 to October 2010. For the retrospective study all the slides available in the department of Pathology, Government Medical College, Jammu which were histologically diagnosed as premalignant or malignant lesions of uterine cervix on cervical biopsies were retrieved from the histopathology archives and screened. In cases where the slide material was not adequate or the quality of the slide was poor, the paraffin blocks were retrieved and fresh slides were prepared for the study. During this period of two years a total no. of 54 cases were reported.
The prospective study included a total no. of 41 cases of premalignant and malignant cervical lesions received in the department of Pathology, Government Medical College, Jammu from November, 2009 to October, 2010. All the chemicals used in this study were purchased by the Pathology department from the Ranbaxy India Limited and Merck Chemicals India. The cervical biopsy specimen were fixed in 10% buffered formalin, dehydrated in ascending grades of alcohol, cleared in xylene and finally embedded in paraffin. 2 - 5 micron thick paraffin sections of uniform thickness were cut on a rotary microtome, dewaxed and stained with hematoxylin and eosin. Hematoxylin and eosin staining was done as follows:
1) After dewaxing in xylene, sections were rehydrated to water by passing through descending grades of alcohol;
2) Stained with Harris Hematoxylin for 10 - 15 min;
3) Rinsed in tap water followed by differentiation in 1% acid alcohol;
4) Sections were washed in tap water until they become blue;
5) Stained with eosin for 2 - 3 min;
6) Dehydrated by passing through ascending grades of alcohol;
7) Cleared in xylene and mounted in DPX (dibutyl phthalate xylene).
A detailed histopathological examination of hematoxylin and eosin stained slides was carried out. Premalignant group of lesions included were squamous metaplasia, mild dysplasia, moderate dysplasia and severe dysplasia (
The hematoxylin-eosin stained slides were screened under 40× objective and 10× eye piece, but the final count of apoptotic cells was carried out under high magnification (oil immersion × 100 lens). From each section, 5 fields devoid of any preservation or fixation artifacts, inflammation and necrosis were selected. Apoptotic cells/bodies in the stroma around the tumours were not counted. In each section, 1000 tumour cells were evaluated for presence of apoptotic cells and Apoptotic bodies. Apoptotic cells with characteristic features of cell shrinkage, condensation and deep eosinophilia of cytoplasm and pyknotic, round to crescentric or irregular nucleus were identified. Apoptotic bodies which typically appear as tiny, round and pyknotic nuclear fragments are seen scattered amongst the tumour cells and occasionally forming small clusters were also identified. Apoptotic index (AI) was calculated as the number of Apoptotic cells and Apoptotic bodies, expressed as percentage of total no. of tumour cells counted in each case. Similarly, mitotic index (MI) was also calculated by counting mitosis among 1000 tumour cells or from dysplastic cells in CIN or from metaplastic lesions in cervical biopsy specimen. Turnover index (TOI) was obtained from sum of MI and AI, i.e., (TOI = MI + AI). After calculating the apop- totic index, mitotic index and turnover index, they were correlated with different grades of the cervical lesions.
The data was analyzed with the help of CDC Epi-Info 7. Mean and standard deviation of apoptotic indices, mitotic indices and turnover indices were calculated for all the subgroups. Fischer’s exact test and Mann Whitney test were used as tests of significance between the sub-groups. Student T-test was used for comparing all the three indices between premalignant and malignant group and also between subgroups of the malignant group.
This study was conducted on 95 cases of various cervical lesions to evaluate apoptotic index (AI), Mitotic index (MI) and TOI (Turn over index) and to correlate these indices with different grades of cervical lesions.
In the present study, age wise analysis after grouping the lesions into premalignant and malignant types revealed that the maximum no. of premalignant lesions were in between the age group of 35 to 54 years (54.5%) and malignant lesions were in the age group 45 - 64 years (69.3%). Histologically, the most common type of premalignant lesion was squamous metaplasia (54.54%) and the most common type of malignant lesion was keratinizing squamous cell carcinoma (69.35%).
For statistical analysis, we divided the premalignant lesions into four subgroups: Subgroup I (Squamous metaplasia vs. mild dysplasia), Subgroup II (mild dysplasia vs. moderate dysplasia), Subgroup III (moderate dysplasia vs. severe dysplasia) and Subgroup IV (Squamous metaplasia vs. moderate/severe dysplasia).
The mean apoptotic indices of premalignant group of lesions, i.e., squamous metaplasia, mild dysplasia, mo- derate dysplasia and severe dysplasia were 0.71 ± 0.3, 0.62 ± 0.6, 1 ± 0.44 and 1 ± 0.6 respectively (
For mitotic index, the mean value for squamous metaplasia, mild dysplasia, moderate dysplasia and severe dysplasia were 0.005 ± 0.02, 0.009 ± 0.08, 0.15 ± 0.05 and 0.25 ± 0.05 respectively (
The turnover indices of squamous metaplasia, mild dysplasia, moderate dysplasia and severe dysplasia were 0.72 ± 0.42, 0.63 ± 0.6, 1 ± 0.44 and 1.25 ± 0.54 respectively (
The mean apoptotic indices of malignant group of tumours, i.e., keratinizing squamous cell carcinoma, non- keratinizing squamous cell carcinoma, adenocarcinoma and adenosquamous carcinoma were 3.38 ± 1.15, 2.92 ± 1.41, 1.76 ± 1.2 and 3.65 ± 0.72 respectively; the mean mitotic indices were 0.57 ± 0.55, 0.42 ± 0.26, 0.2 ± 0.07 and 0.2 ± 0.04 respectively and the mean turnover indices were 3.96 ± 1.38, 3.27 ± 1.5, 1.96 ± 0.9 and 3.85 ± 0.42 respectively (
Within the malignant group, we compared well differentiated (keratinizing squamous cell carcinoma) with less well differentiated groups (non-keratinizing squamous cell carcinoma, adenocarcinoma and adenosquamous carcinoma). Statistical analysis revealed a significant difference in TOI between the two subgroups of malignant tumours (
Diagnosis | No. of cases | Apoptotic index (mean ± SD) | Mitotic index (mean ± SD) | Turnover index (mean ± SD) |
---|---|---|---|---|
Squamous metaplasia | 18 | 0.71 ± 0.3 | 0.005 ± 0.02 | 0.72 ± 0.42 |
Mild dysplasia | 11 | 0.62 ± 0.6 | 0.009 ± 0.08 | 0.63 ± 0.6 |
Moderate dysplasia | 2 | 1 ± 0.44 | 0.15 ± 0.05 | 1 ± 0.44 |
Severe dysplasia | 2 | 1 ± 0.06 | 0.25 ± 0.05 | 1.25 ± 0.54 |
Keratinizing squamous cell carcinoma | 43 | 3.38 ± 1.15 | 0.57 ± 0.55 | 3.96 ± 1.38 |
Non-keratinizing squamous cell carcinoma | 14 | 2.92 ± 1.41 | 0.42 ± 0.26 | 3.27 ± 1.5 |
Adenocarcinoma | 3 | 1.76 ± 1.2 | 0.2 ± 0.07 | 1.96 ± 0.9 |
Adenosquamous carcinoma | 2 | 3.65 ± 0.72 | 0.2 ± 0.04 | 3.85 ± 0.42 |
Squamous metaplasia vs. Mild dysplasia | Mild dysplasia vs. Moderate dysplasia | Moderate dysplasia vs. Severe dysplasia | Squamous metaplasia vs. Mod/Severe dysplasia | |
---|---|---|---|---|
Apoptotic index | 0.669 | 0.447 | 0.102†† | 0.973 |
Mitotic index | 1† | 0.03*† | 0.102†† | 0.0005*† |
Turnover index | 0.69 | 0.473 | 1†† | 0.519 |
†Fisher’s exact test; ††Mann Whitney test; *Significant.
Premalignant vs. Malignant | Well differentiated vs. Less differentiated | |
---|---|---|
Apoptotic index | 0.0001* | 0.111 |
Mitotic index | 0.0001* | 0.128 |
Turnover index | 0.0001* | 0.04* |
**Highly significant; *Significant.
Statistical comparison between premalignant and malignant group of lesions showed a highly significant difference between the AI, MI and TOI of premalignant group of lesions and malignant group of lesions with a p value of p = 0.0001 (
Cervix is commonly affected with various pathological conditions like cervicitis, hyperplasia, metaplasia, dysplasia, benign tumours and malignant neoplasms. Squamous cell carcinomas irrespective of the site, evolves through a sequence of changes occurring in the normal squamous epithelium, ranging from dysplasia of varying degrees to frank malignancy [
Biological behavior of the tumour depends not only on its proliferative activity but also on the number of cells dying either by apoptosis or by ischemic necrosis. Proliferative activity of a tumour depends on stimulatory growth signals from various oncogenes and may be accompanied by inactivation of one or more tumour suppressor genes [
Although apoptosis has been evaluated & shown to correlate with tumor grade and subtype in some malignant lesions including those of large intestine, endometrium, prostate, breast [
The present study was conducted on 95 cases of various cervical lesions to evaluate apoptotic index (AI), Mitotic index (MI) and TOI (Turn over index) and to correlate these indices with different grades of cervical lesions. In our study, maximum no. of premalignant lesions was in the age group of 35 to 54 years (54.5%) and malignant lesions were in the age group 45 - 64 years (69.3%). Vijaya et al. 2008 reported maximum no. of premalignant lesions between 35 - 54 years (61.1%) and malignant lesions were in age group 35 - 64 years (80.2%).
In the present study, majority of the lesions were malignant 62 cases (65.2%) and 33 cases (34.73%) were premalignant. Sagol, O., et al., 1999 conducted study on 55 (64.7%) premalignant and 30 (35.2%) malignant lesions (squamous cell carcinoma of cervix) [
Histologically, the most common type of premalignant lesion was squamous metaplasia (54.54%) which is similar to results obtained by Vijaya, et al., 2008 who reported the most common type of premalignant lesions as squamous metaplasia (66.9%). The most common type of malignant lesion in our study was keratinizing squa- mous cell carcinoma (69.35%) followed by non-keratinizing squamous cell carcinoma (22.58%).
In our study, comparison between the mean AI and mean TOI in Group I (squamous metaplasia vs. mild dysplasia) showed statistically insignificant difference (
Present study shows that mean AI, MI and TOI in Group II (mild dysplasia vs. moderate dysplasia) and Group III (moderate dysplasia vs. severe dysplasia) increases with increasing rate, i.e., from mild dysplasia to moderate dysplasia to severe dysplasia. On statistical analysis mean AI and TOI is found to be statistically insignificant. This insignificant difference could be attributed to the lack in the number of cases in moderate and severe dysplasia, as our study involves both retrospective and prospective analysis and maximum number of patients reported with carcinoma cervix. Mean MI in Group II (mild dysplasia vs. moderate dysplasia) was found to be statistically significant (p = 0.03) (
Vijaya, et al. (2008) reported that mean AI, MI increases with the severity of dysplasia in premalignant lesion. The difference was statistically significant (p < 0.01) in Group II (mild vs. moderate dysplasia) and Group III (moderate vs. severe dysplasia). Sagol, O., et al. (1999) did not find any statistically significant difference between CIN I and CIN II as well as between CIN II and CIN III groups in terms of apoptotic and mitotic cell counts. Mitotic cell counts were found statistically higher in CIN III group when compared with CIN I and CIN II groups together coinciding with the results of our study in Group II (mild vs. moderate dysplasia) and Group III (moderate vs. severe dysplasia). Pahuja, S., et al. (2003) reported cell proliferation by AgNOR count in vari- ous grades of pre-invasive cervical epithelial lesion and showed progressive rise in their mean value with increas- ing grade of pre-invasive lesion [
In the present study, we have done the additional statistical analysis for Group IV (Squamous metaplasia vs. moderate/severe dysplasias) and found that mean AI and TOI was statistically insignificant but mean MI is shown to be significant statistically (p = 0.0005). This shows that number of mitosis increases with increasing grade of lesion.
We have also compared the mean AI, MI and TOI of malignant lesion as a whole group with the premalignant lesion and found that the AI, MI and TOI increased as the nature of lesion changed from premalignant to malig- nant. This difference between both the groups was very highly significant (p = 0.0001) (
Vijaya et al. (2008) also reported significant difference (p < 0.01) in mean AI and MI with increasing rates of lesion from dysplasia to carcinoma cervix. Dey, P., et al. (2000) reported that mean AI (p = 0.0028), MI (p = 0.0018) and TOI (p = 0.0014) increases from lower to higher grades of CIN to carcinoma of cervix. The difference was found to be highly significant correlating our results with their study. Sagol, et al. (1999) also reported that the squamous cell carcinoma cervix group showed significantly higher mitotic and apoptotic cell counts when compared with pre-neoplastic lesions. They concluded that both apoptosis and mitosis are markedly increased in progression towards malignancy in cervical epithelium. Mitotic cell counts may be helpful in predicting the extent of the disease in squamous cell carcinoma cervix
In the present study, we further subdivided the malignant lesions into well differentiated (KSCC) and less well differentiated (NKSCC, adenocarcinoma, adenosquamous carcinoma lesions) and compared the AI, MI and TOI. There was no statistically significant difference between the AI and MI of both the groups. The difference in TOI was however statistically significant (p = 0.04) (
Our results correlate with Sagol, O., et al. (1999) who also reported that there was no statistically significant difference in the apoptotic and mitotic cell counts between non-keratinizing and keratinizing squamous cell car- cinoma. They also showed that in the squamous cell carcinoma group, apoptotic cell counts did not show sig- nificant difference between tumour stages but mitotic counts were significantly higher in advanced stage tu- mours.
To conclude, the apoptotic cells can be easily demonstrated in routine H & E stained sections, though a high degree of variability still exist in the apoptotic index reported by various authors. To avoid this inter-observer variability the established criteria for recognition and counting of apoptotic cells should be strictly adhered so that the cervical lesions can be characterized properly according to their potential for invasiveness.
We conclude that apoptotic cells and mitosis can be readily and accurately demonstrated on routine H & E stained sections. AI and MI are the simplest techniques that can be employed in any laboratory. Proliferative and apoptotic indices have been found useful in distinguishing between benign and malignant lesions of the cervix. Observed increase in apoptotic cell with the grade of the lesion suggests a mechanism whereby apoptosis helps to eliminate cells that have been produced in excess.
We also conclude that proliferative activity of a lesion is a reliable indicator of its malignant potential and together with apoptotic count gives an idea about the net growth of a tumour.
Conflict of Interest
Nil.
AI = Apoptotic Index;
MI = Mitotic Index (MI);
TOI = Turnover Index;
HPV = Human Papillomavirus;
H & E = Hematoxylin and Eosin;
CIN = Cervical Intraepithelial Neoplasia;
ISEL = In Situ End Labeling;
TUNNEL = TdT Mediated dUTP-Biotin Nick End Labeling;
KSCC = Keratinizing Squamous Cell Carcinoma;
NKSCC = Non-Keratinizing Squamous Cell Carcinoma.