European Journal of Cardiovascular Medicine

Cervical cancer is a major global public health problem. With 528,000 new cases every year, cervical cancer is the fourth most common cancer and cause of cancer death (266,000 deaths in 2012) in women worldwide accounting for 7.5% of all female cancer deaths. Almost nine out of 10(87%) of cervical cancer deaths occur in less developed countries and more than one fifth of all new cases are diagnosed in India. Cervical cancer is 2nd most common malignancy after breast carcinoma among Indians.¹ According to IARC estimates, mortality from cervical cancer is expected to witness a 79% increase from 74,118 deaths in 2002 to 132,745 deaths by 2025.² This large scale morbidity and mortality due to cervical cancer is totally unwarranted not only because the definitive cause of cervical cancer is now known, but also because the disease takes a long time to develop after initial infection with high risk Human papilloma virus (HPV).³

Incidence of cervical cancer can be decreased by regular screening and treatment of precancerous lesions. Although Pap smear is central to screening, it has some limitations, most important being its limited sensitivity which is between 47-62% and the subjective interpretation of the results.⁴ Each year, 330.000 new Cervical Intraepithelial Neoplasia (CIN) cases occur in the European Union, with about a half of them diagnosed as CIN I.⁵ CIN is usually a long pre-invasive process, characterized microscopically as a range of events progressing from cellular atypia to various grades of dysplasia, which over time can progress to invasive cervical carcinoma.⁶

The new WHO classification applied LSIL (Low-grade Squamous Intraepithelial Lesion) and HSIL (High- grade Squamous Intraepithelial Lesion) terminology.⁶ However, previous classifications classified CIN as CIN I, CIN II and CIN III based on the degree of dysplasia.⁷ Accurate histological grading of CIN lesions was important for clinical management of patients, because CIN lesions were monitored and treated differently. CIN I was usually regarded as benign and no therapy was indicated, because it regresses in about 80% of cases.⁸

The aim of the present study was to compare the expression of p53 and Ki-67 in cervical intraepithelial neoplasia and different histological types and grade of cervical cancer.

The study was an observational study conducted during a period of one and half years in the Department of Pathology and Department of Obstetrics and Gynaecology, Government Medical College and Rajindra Hospital, Patiala, Punjab. 100 cases were included in the study.

INCLUSION CRITERIA:

• Histopathologically diagnosed biopsy of cervix and hysterectomy specimens for cervical intraepithelial neoplasia and carcinoma cervix.

EXCLUSION CRITERIA:

Cervical biopsies with active infections, chronic inflammatory disease, and histopathologically undetermined cervical abnormalities.

Histopathological Examination: Cervical biopsies and hysterectomy specimens were subjected to routine processing for paraffin embedding four-to-five-micron thick sections were taken from paraffin embedded blocks and stained with hematoxylin and eosin (H & E) stain.

 Cervical tumors were then typed according to the WHO classification system. The modified Broder’s grading system was used to grade the tumors. The important microscopic subtypes of squamous cell carcinoma, presence of additional in-situ component and pattern of infiltrative margins of invasive component were studied. IHC was performed on p53 and Ki-67.

PROCESSING OF TISSUE SECTIONS:

• Dehydration was carried out by passing the tissue sections through a series of ascending grades of ethyl alcohol 50%, 70%, 90% and absolute alcohol.
• Clearing was done by passing the tissue through three cycles of xylene of one and half hour duration each
• Impregnation with molten wax was done.
• It was followed by embedding in paraffin wax.
• After that tissue was cut into 3 microns thick sections and was stained with hematoxylin and eosin stain.

  HEMATOXYLIN AND EOSIN STAINING PROCEDURE: -

• Slides were kept on a hot plate in order to dewax the sections at a temperature up to 60 degrees.
• Slides were deparaffinized in xylene then graded alcohol to water.
• Then, it was stained in hematoxylin for 15 minutes.
• The sections were washed well in running tap water until sections turn blue.
• The sections were then differentiated in (1% HCL in 70% alcohol) for 5-10 seconds and it was washed with tap water for 5 min or less.
• Then it was stained with 1% eosin for 10 min and followed by washing in running tap water for 1-5 min.
• In last, sections were dehydrated once again through graded alcohol cleared in xylene and mounted with DPX avoiding air bubbles.

  IMMUNOHISTOCHEMISTRY: - p53: - Monoclonal Mouse BP-53-12 was applied on three microns thick, formalin based, paraffin embedded tissue sections.

Ki-67: - Monoclonal mouse Ki-67-30-9 was applied on three microns thick, formalin fixed, paraffin embedded tissue sections.

Control: - Sections from normal tonsil tissue and breast cancer tissue was used as a positive control.

 PROCEDURE: -

1. Backing of slides in hot air oven.
2. Deparaffinization + Dehydration + Antigen retrieval in high ph (Blue) solution for 30 minutes at 95℃.
3. Wash with distilled water for 3 minutes each applying 3 changes.
4. Apply Peroxidase Block solution for 30 minutes in humid chamber.
5. Wash with wash buffer for 3 minutes each applying 3 changes.
6. Apply Primary Antibody for 1 hour in humid chamber.
7. Wash with wash buffer for 3 minutes each applying 3 changes.
8. Apply Secondary Antibody for 30 minutes in humid chamber.
9. Wash with wash buffer for 3 minutes each applying 3 changes.
10. Apply DAB solution wait till development of brown color for control tissue.
11. Wash with distilled water.
12. Dehydrate with 50%, 80%, 100% alcohol.
13. Apply Xylene, DPX, Coverslip for mounting.

Ki-67 and P53 SCORING BY IMMUNOHISTOCHEMISTRY: -

GRADING OF INTENSITY OF STAINING OF p53 AND Ki-67 ⁹: -

PERCENTAGE OF POSITIVITY OF p53 AND Ki-67 STAINING ⁹:-

Statistical analysis was done to find immunohistochemical expression of p53 and Ki-67 by using Chi-Square and Anova test.  

Table 1: Baseline characteristics

In present study, we found that most of the patients were in 41-60 years of age group. In present study we found mean age was 53.22 ± 11.748 years. Minimum age was 32 years while maximum age was 82 years. Range of age was 50 years. In present study we have 49 % cases in post-menopausal state, while 29 (29%) cases in Peri-menopausal 22 (22%) cases in Pre-menopausal state. In present study we found that 88% cases had OCP/HRT while 12% cases had no history of OCP/HRT. P-value was 0.00001 which was significant. Most of the patients had parity between 3-5 and showing 77% positivity followed by >5 showing 18% and the least number of patients showing 5%.

Table 2:  Gross Examination of Cervical Growth, tumor type and histological grading

This study showed that 75% of patients showing ulceroproliferative growth which is an exophytic growth in cervix that completely replaces cervix.  This was followed by 16% of patients showing nodular growth, 4% showing papillary growth and 3% showing polypoidal growth. In present study we found that squamous cell carcinoma was most common cervical neoplasia seen in 92% of cases, while adenocarcinoma was second most common type seen in 4% cases. In present study we found that moderately differentiated was seen in 82(89.1%) of cases, while poorly differentiated was seen in 3 (3.3%) of cases and 7 (7.6%) were well differentiated.

Table 3: Age wise distribution of Squamous cell carcinoma, adenocarcinoma

Among SCC cases we found that 14 (15.21%) belongs to 20-40 years, 54 (58.69%) belongs 41-60 years, 17 (18.47%) belongs to 61-70 while 7 (7.60%) were above 70 years. Youngest age was 32 years and eldest age was 82 years. Among Adenocarcinoma cases we found that 1 (25%) belongs to 20-40 years, 1 (25%) belongs 41-60 years, 2 (50%) belongs to 61-70 while 0 (0%) were above 70 years.  In this study, 4 patients were of age 67 and 65 years followed by 46 and 33 years old. Maximum patients were in the age group of 61-70 followed by 41-60,21-40 and >70 there were no patient. Among CIN cases we found that 1 (25%) belongs to 20-40 years, 2 (50%) belongs 41-60 years, 1 (25%) belongs to 61-70 while 0 (0%) were above 70 years.  In this study, 4 patients were of age 61,60,45 and followed by 40 years old. Maximum patients were in the age group of 41-60 (50%) followed by 61-70 (25%), 21-40 (25%) and in >70 there were no patients.

Table 4: Intensity and percentage of staining of p53 of SCC

Among SCC cases, we found 57 (61.95%) had grade 3, 20(21.73%) had grade 0, 14(15.21%) had grade 2 and   1(1.08%) had grade 1. In this study, according to age, patients in the age group of 40-60 had grade 3. In present study, most of patients had p53 percentage positivity more than 60%.  In this study, patients in the age group 40-60 had >60% positivity.

Table 5: Intensity and percentage of staining of Ki-67 in Squamous cell carcinoma

Among SCC cases we found that 8 (8.69%) had grade 0, 7 (7.60%) had grade 1, 7 (7.60%) had grade 2 while 70 (76.08%) had grade 3. In present study, patients of age group 40-60 had grade 3. In present study, most of patients had Ki-67 percentage positivity more than 60%.  In this study, patients in the age group 40-60 had >60% positivity.

Table 6: Intensity and percentage of staining of Ki-67 in Adenocarcinoma

Among adenocarcinoma 3 (75%) had grade 3 while 1 (25%) had grade 2. According to age, 3 patients of having age 67, 33 and 46years had grade 3 while one of patient having age 65 years had grade 2. Among Adenocarcinoma, all patients had Ki-67 above 60%. According to age, one of the patients having age 46 years had 97% positivity followed by 65 years had 88%,67 years had 87% and 33 years had 56% positivity.

Table 7: Intensity and percentage of staining of p53 in CIN

Among CIN cases we found 3 (75%) cases were grade 3. In present study according to age 3 patients having age of 65,40 and 45 years had grade 3 followed by 61 years of age had grade 2. Among CIN all patients had >60% positivity. In present study according to age one of the patients having age of 40 years had 96% positivity followed by 45 years had 87% ,60 years had 75% and 61 years had 70% positivity.

Table 8: Intensity and percentage of staining of Ki-67 in CIN

Among CIN cases we found 4 (100%) cases were grade 3. According to age, patients of having age 61,60,45 and 40 years had grade 3. All patients had Ki-67 percentage above 60%. According to age,3 patients of having age 60,45 and 40 yrs had 85% positivity followed by a patient of having age 61 years had 78% positivity.

Table 9: COMPARISION OF p53 AND Ki-67 AMONG SCC

We found that Ki-67 and p53 were significantly positivity among SCC cases.

Fig. 1: Grade 5 positivity (Intensity-2 , percentage-85%, score-5) of p53 expression in moderately differentiated squamous cell carcinoma

Fig. 2: Grade 5 positivity(Intensity- 2, Percentage- 66%, Score-5)of Ki-67 expression in moderately differentiated squamous cell carcinoma

Fig. 3: Grade 2 positivity of p53 expression in CINIII

Fig. 4: Diffuse positivity of Ki-67 in CINIII

Cervical cancer is the second commonest cancer in the world and is a major cause of morbidity and mortality. Carcinoma of cervix is the leading cancer in India, common in females between 15 and 44 years of age. It is unique among human cancers in that it was the first to be virtually solely attributed to an infectious agent.¹⁰ It is one of the most commonly reported gynecological malignancies. Although human papilloma virus (HPV) is considered as the one of the prime factors in the development of cancer but several studies have reported that there is an association between genetic polymorphisms and environmental factors.¹¹ In early stages of cervical cancer, this is likely to be cured by a combined treatment of surgery, radiotherapy or chemotherapy. In contrast, options for patients in late stages involve alternative treatment such as chemoradiotherapy with poor prognosis. So, routine pap smears and HPV testing have largely improved outcome of cervical cancer in developed countries.¹²

In present study, there are 100 cases. The youngest age was 32 years and oldest age was 82 years. Range of age was 50 years. The peak age was 40-60 years which was in concordance with the study done by Jain S at el¹³ which showed that maximum number of patients belong to 45-55 years of age and Tan G C et al¹⁴ which showed mean age 43.8 years. In present study, most of the patients were post-menopausal. In the study of Arbyn M et al¹⁵ showed that cervical cancer incidence was more common in menopausal women after 50 years of age. In present study, most common cervical cancer was squamous cell carcinoma. Out of 100 patients, 92 cases were of squamous cell carcinoma followed by 4 cases of adenocarcinoma and 4 cases of CIN which is in concordance with the studies done by Raju K et al¹⁶ and Patel R et al.¹⁷

In our study, most of the patients had parity between 3-5 which showed 77% of patients.  The study of Tekalegyn Y et al¹⁸ also explained that developing cervical cancer was more than two times higher among the women with high parity (>3) compared to their counterpart. In present study, moderately differentiated was seen in 82(89.1%) cases , while well differentiated was seen in 7(7.6%) cases and poorly differentiated was seen in 3(3%) cases which was in concordance with study done by Jain S et al¹³ and  Raju K et al.¹⁶  In present study most of patients were in age group of 41-60 years and mean age was 53.22 years which is similar with studies done by Patel R et al¹⁷ and Jain M A et al.¹⁹ In present study, 92 cases were enrolled, 57(61.95 %) cases had grade 3 which is in concordance with other studies done by Raju K et al¹⁶ and Jain S et al.¹³  There were 92 cases in present study in which 70(76.08%) had grade 3 which is in concordance with the study done by Raju K et al.¹⁶

In conclusion, p53 and Ki-67 expressions were directly associated with severity of cervical lesions. The highest expression of both markers was found in invasive carcinoma and CIN2/3 and lower in descending order for CIN 1. The significant differences in these markers’ expression may be useful in equivocal histologic features among the cervical intraepithelial lesions.  So, our study indicates that p53 and Ki-67 are powerful prognostic markers.

 In future, it is necessary to carry out studies with large samples of carcinoma cervix and correlating p53 and Ki-67 markers with the clinicopathological parameters with follow up of patients. This will provide an opportunity for development of p53 and Ki-67 targeted therapy for carcinoma cervix. Few cases of carcinoma cervix were p53 and Ki-67 negative suggesting that the others factors may be involved in carcinogenesis.

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