INTRODUCTION cancer patients. OBJECTIVE The main objectivesINTRODUCTION cancer patients. OBJECTIVE The main objectives

INTRODUCTION

Cervical
cancer is the third-most commonly diagnosed cancer and the fourth leading cause
of cancer death in females worldwide (1). India accounts for 27% (77,100) of
the total cervical cancer deaths (2). The estimated age standardized incidence
rate was 22.9/100,000 women. (3) In India, the incidence of cervical cancer
significantly rises around the age of 45 years and peaks at 55 years of age. Despite
advances in surgical capabilities and chemotherapy strategies, a substantial
proportion of patients still die from recurrent or chemoresistant disease.

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Cancer stem
cells (CSCs), a small subset of tumor cells, are characterized by self-renewing
capacity, heterogeneity and re­sistance to several therapies (such as
chemotherapy and radio­therapy), and contribute to the process of tumor
development, infiltration, metastasis and recurrence (4).  It is postulated that
CSCs may originate from normal stem cells and
express the same cell markers as normal stem
cells. Such a model has been proposed for cervical CSC
markers as well 5. Among CSC markers, octamer-binding
transcription factor 4 (OCT4) and sex determining region Y-box 2 (SOX2) are
transcriptional factors involved in the regulation of several target genes.
OCT4 belongs to the POU (Pit-Oct-Unc) transcriptional factor family and plays a
key role in stem cell pluripotency and differentiation by determining the fate
of embryonic stem cells 6. Prior studies suggest OCT4 and SOX2
have a key role of tumorigenesis and prognosis of cancer. However, the prognostic
significance of OCT4 and SOX2 is not clearly defined in cervical premalignant
and malignant lesion.

Currently, there is a
lack of biomarkers for predicting the response to chemotherapy in cervical
cancer patients. The present has, thus, been planned accordingly in which the purpose is to investigate the association of cancer
stem-cell marker OCT4 expression with clinicopathological features and predict the
prognosis in cervical cancer patients.

OBJECTIVE

The main objectives of
this study will be:

1.     
To register clinically diagnosed cases
of cervical cancer attending the OPD of Department of Gynaecology and
Obstetrics at Era’s Lucknow Medical College and Hospital

2.     
To stage clinically and
histopatologically grade the disease

3.     
To stain for OCT4 stem cell marker by
immunohistochemistry

4.     
To correlate the above clinicopathologically

METHODOLOGY

Study
Area: Era’s Lucknow Medical College and Hospital.

Clinical Subjects:
A written informed consent will be taken from all participants before
collecting their tissue samples. Tissue samples of 20 subjects including 10
patients of clinically diagnosed cervical cancer as cases , and 10 patients of
chronic cervicitis as controls will be collected from the Gynaecology OPD of
the Department of Obestetrics and Gynaecology at Era’s Lucknow Medical College
& Hospital, Lucknow. Since this is a time-limited study with financial
constraints, the number of cases has been arbitrarily kept at 20 and has not
been calculated according to any formula. Medical records will be obtained to
review patient data including age, cancer stage, tumor differentiation, cell
type, tumor size, lymphovascular space invasion (LVSI) and lymph node (LN)
metastasis.

Study design: Case
control study

Inclusion criteria

·        
All fresh cases who have been clinically
diagnosed with cervical cancer or cervical hyperplasia who gave consent for
participation in the study will be included.

Exclusion criteria

·        
Patients who have any history of second
malignancy in either present or past.

·        
Any patient who has any immunodeficiency
disease will not be included in the study.

Biopsy will be taken and fixed in
formalin. Tissue will be processed in automatic histokinette and 4 µ thick
sections will be cut and stained in Haematoxylin and Eosin. Sections will be
examined under the guidance of an able pathologist and grading of the disease
will be done.

Immunohistochemistry

Cancer
stem cells (CSC) will be investigated in tissue biopsy by Immunohistochemistry
at histopathological level by commercially available markers (DAKO), for;

–         
OCT4

Protocol
for Immunohistochemistry:

1.      Bring
section down to water:

De wax the
section by three changes of xylene, absolute alcohol, 90%, 70% and 50%, each
for 3 min.

2.
Retrieval:

·        
Keep the slide in antigen retrieval in
coplin jar; place the jar into the pressure cooker on hot plate. After one
whistle, switch off the plate and wait untill all of the pressure is released.

·        
Flood the pressure cooker. Do not remove
the slides until cool.

·        
Wash slides with 3 – changes of
distilled water (3 mins)

·        
Wash slides with 3- changes of tris
buffer.

·        
Apply primary antibody in moist chamber
for 1 hr. (Room Temp.)

·        
Wash slides with 3- changes of tris
buffer.

·        
Apply secondary antibody for 30 mins. (
Room Temperature)

·        
Again wash with 3- changes of tris
buffer.

·        
Apply DAB chromogen solution for 5- 10
mins.

·        
Observe the colour under microscope. When
the colour developed (Khaki colour), place the slide in running tap water for
5- mins.

·        
Counterstain in hematoxylin for 1 min.

·        
Wash slides in running water.

·        
Bluing with Scott mixture.

·        
Dehydrate, clear, and mount.

Positively expressed CSCs will be
counted in 10 different High power fields (400X) to calculate the numbers of
cells present/Sq.mm. The findings will be correlated with the clinical stage of
the disease, and with histopathological findings. The findings of Cervical
Cancer cases will be compared with those of control. Any metastasis if
clinically detected and if possible will be investigated histopathologically.

IMPLICATIONS

If the study indicates
overexpression of OCT4 in high grade cervical cancer, it shall be taken as a
marker for poor prognosis. Therefore, this data might provide valuable
information on the nature and behavior of tumors, leading to a new strategy for
targeting CSCs and perhaps one step closer to the prevention of cancer
recurrence and metastasis.

REFERENCES

1.     
Jemal A, Bray F, Center
M. Global cancer statistics. CA: A Cancer Journal for Clinicians.
2011;61(2):69-90.

2.     
Mathew A, George PS. Trends in incidence
and mortality rates of squamous cell carcinoma and adenocarcinoma of cervix–
worldwide. Asian Pac J Cancer Prev. 2009; 10:645-650.

3.     
International Agency for Cancer, World
Health Organization. Globocan 2012: Estimated Cancer Incidence, Mortality and
Prevalence Worldwide in 2012. India Cancer Cervix Incidence and Mortality
Estimates. Lyon; 2012.

4.     
Beck B, Blanpain C.
Unravelling cancer stem cell potential. Nature Reviews Cancer.
2013;13(10):727-738.

5.     
Takebe N, Ivy S.
Controversies in Cancer Stem Cells: Targeting Embryonic Signaling Pathways.
Clinical Cancer Research. 2010;16(12):3106-3112.

6.     
Schöler H, Ruppert S,
Suzuki N. New type of POU domain in germ line-specific protein Oct-4. Nature.
1990;344(6265):435-439.