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Cancer is a group of diseases that cause cells in the body to change and grow out of control. These cells have the ability to spread, either by direct growth into adjacent tissue through invasion, or by implantation into distant sites by metastasis. Cancer diseases are different in type, and behave differently to each other. One of its kinds is cervical cancer, which is the cancer of the cervix in women.
The cervix is known as the neck of the womb, and is located in the lower part of the uterus, connecting the body of uterus to the vagina. Most cases of cervical cancer occur where the endocervical part (the closest part to the body of uterus) meets with the ectocervix (the part close to the vagina). This area is known as the transformation zone as illustrated in fig.1.
Diagram showing the transformation zone on the cervix
Figure 1: The position of the cervix.
Cervical cancer seems to be on the rise. In developing nations like in Eastern Africa, cervical cancer has increased drastically (Dyess, 2008). And while in the world cervical cancer is declining, it is still the world's second most common disease and third leading cause of deaths among women (Dawn). According to the UK cancer research records, about 2,800 individuals are diagnosed with cervical cancer every year. After Jane Goody, a noted reality television star, got diagnosed with cervical cancer, more women were diagnosed in the UK with cervical cancer thanks to a 12% increase in screening, the first such increase since 2002 (Cancer Research UK, 2009; Sturcke, 2009).
Cervical cancer is associated with number of risk factors that increases the percentage of developing the disease. These risk factors include HPV infection. This is the most common cause of cervical cancer in women but not always, as well as other viral infection (HIV, bacterial infection (Chlamydia), smoking and oral contraceptive pills or hormones. For the diagnosis of cervical cancer, specific biomarkers are detected. These biomarkers can confirm the exciting or absence of the cervical cancer pre-cursors. These markers include P16INK4A, which is a direct marker related to the presence of HPV and is associated with the disease progression, thus; it provides straight-forward and precise prediction of the cervical lesions. Other biomarkers which are used in cervical detection and diagnoses also include Ki-67 and E-Cadherin. Ki-67 is a marker that can be found in most cell-cycle phases apart from phase G0. Once the cell cycle is distributed through the binding of the HPV DNA E6 and E7, the protein is abnormally expressed. The amount of this marker detected can demonstrate the degree of maglinancy. Monhoz et al, (2009), adopted from Dursan et al, stated that a reduction of E-Cadherin expression is related to cancer progression and its aggressive behaviour, serving an indicator of the malignancy potential in cervical cancer.
In cytology laboratory, a smear from the ectocervix is obtained and tested by looking at the morphology of the cervical cells, detecting any alterations to the cells and/or pre-malignant and malignant conditions including cervical intra-epithelial neoplasia (CIN) and cervical dysplasia. These screening tests include papanicolou and kreyberg stains which allow an early diagnosis of the disease.
Papanicolou stain is the standard technique used for cervical smears due to its reliability, simplicity and effective means detecting any alterations in the cervix wall by microscopical examination. This staining method enhances the differentiation between cellular and non-cellular substances such as acidophilic, basophilic, fibrins or pigments, plus to its ability in staining fungi and bacteria.
To perform a pap stain, cells sample is collected from the cervix using speculum to widen the vagina for viewing of the cervix, a plastic spatula and small brush to collect cells. This technique is known as scraping. The cells are fixed in a 95% ethanol solution (0.5% acetic acid and 10% PEG) for protection till it reaches the laboratory for analysis. In the laboratory, the cytotechnologist washes the slides in 50% isopropanol for two minutes to remove any PEG so no artefacts will occur during staining. The method is carried on straight after so the smears not allowed drying. Starting from washing the slides in deionised water for 1 min, stained in Harris's haematoxylin for 5mins and followed by rinsing in deionised water (only for 30 secs). Enough amount of 0.5% aqueous HCI is placed on the slide and left 10-20 sec till the smear produce salmon colour then wash off with deionised water (30 secs). The slide is then dipped in Scott's alkaline tap water solution for 1 min and then transferred to deionised water for washing for 2 minutes. Washing in alcohol series is then followed ( 50%, 70%, 95% X 2) 1 min each and then 10 minutes stain in orange G6 followed by washing step for 30 sec in 95% isopropanol. Slide stained in Gill's eosine-azure solution for 5 minutes and washed in two changes of 95% alcohol, 1 minute each. And finally incubated in clearing agent for 5 minutes then covered with DPX and coverslip. The cells are now ready for critical examination of the nuclei and cytoplasmic components.
Tumour cells from cervical sample can be identified also by the use of Kreyberg's staining method, which allows distinguishing CIN from adenocarcinoma due to the modification of other methods too. Modified Kreyberg's staining allows the detection of HPV infected cells (warty), as it stain acid mucin, prekeratin and keratin at once. The procedure of this technique is to rehydrate the sections through alcohol and stain with haematoxylin for 5 minutes followed by washing step in water. HCI is used if necessary for differentiation of the sections for maximum of 30 seconds. Sections is dipped in blue warm water, and rinsed afterward in distilled water. Stained in phloxine for 3 minutes then washed in water to remove any excess stain followed by wash in distilled water. Alcian blue is added to the slide cover all sections for 8 minutes and washed off in distilled water, and stained in orange G for 13 minutes and quickly dehydrated in 95% alcohol followed by two changes of alcohol, clear and mounted. Under the microscope, the use of pholxine stained the acid mucin blue/ green, while orange G stained prekeratin and keratin orange to red orange. Nuclei appear brown under the microscope and the sytoplasm grey to brownish colour.
Most cases of cervical cancer are related to previous HPV infection, however not all infections develop cervical cancer. Many studies including Munhoz et al, 2009 and Wang et al, 2005, stated that HPV encodes an important protein that plays a big role in controlling and inactivating of the Rb and p53 tumour suppressor genes through the interaction between its oncoproteins E6 and E7. These are P16NK4A and P14ARF and are considered as the status to distinguish the non-HPV related cancers from others by immunohistochemistry. This is because p16NK4A is overexpressed predominantly in the nuclear and cytoplasm of most of the tumour samples and are considered being positive. Also, the expression of other proteins such as Ki-67 and E-cadherin is respectively related to p16, and can be detected as a prognosis factor (Munhoz et al, 2005). The photomicrograph below (fig.2) shows how can be p16 useful for the diagnosis of cervical cancer. (a) Shows normal cells, with no expression of p16. (b) Is to be class CIN I, where p16 is detected compared to (a). (c) CIN II, and (d) CIN III where a large amount of p16 is expressed.
Figure 2: P16 antibody expression in normal and Patient samples in an endo-cervical cancer.
The cervical surface is covered with a layer of epithelium cells, arising from the stratified squamous epithelium. This epithelium is produced in the basal layer, maturing in the middle layer and becoming shed from the superficial layer. These superficial cells are large in size (~50-60um). When stained, the nuclear become very condense. In most smears, this is the most common seen cells. When stained in pap, they appear pink to orange and are often angular or squared in shape (Fig.3 - [A]). Intermediate cells are smaller cells (~40-50um) compared to the superficial cells and tends to have a larger nucleus with fine granular chromatin. The cell cytoplasm is pale green in pap stain as shown in the photomicrograph fig. 3 [B]. [C] Are the parabasal cells. They are smaller than the intermediate cells, around 20-30um with a larger central nucleus containing coarser chromatin. These cells are detected in small numbers always; however they increase when the female reach late 40's or early 50's (Menopause). The cells are rounded. In pap stain, the cytoplasim is green. Few polymorph neutrophils are seen with lobed nuclei [D].
Figure 3: Morphology of normal cervical cells (day 14-21 during cell cycle).
Any alteration to the epithelium cells in the cervix are referred to as cervical intraepithelial neoplasia (CIN). In some cases, CIN are progress to invasive cancer. These changes i.e. CIN, may begin as low grade CIN and develop a higher grade CIN III (Fig. 5). These changes are classified as follows:
CIN I: Mild dysplasia,
CIN II: Moderate dysplasia,
CIN III: severe dysplasia / carcinoma in situ.
Figure 4: The biology of cervical cancer. (Over a number of years, CIN is known to be causative of cervical cancer).
Normal cells of the cervix are round in shape and young at the bottom layer, once mature, they move to the surface and flatten out. In cervical dysplasia, the cells lack this organisation of the cells and. The mild dysplasia (CIN I) minor cells are abnormal and is characterised by the koilocytotic atypia in the superficial layer of the epithelium. The dysplasia in the moderate phase (CIN II) is in a rise. Mature keratinocytes are delayed into the middle third of the epithelium. Cell size and nuclear vary in CIN II. Basal cells occupy in the lower half, extending to the middle third of the epithelium. In CIN III or cancinoma in situ, the event is sever and observed by the large number of variation in cells and nuclear size. Cells are immature in the middle layer with big sized nucleus. Abnormal mitosis is detected. However, abnormal cells have not reached the underlying tissues. If so; that would be referred to as invasive cancer.
Table 1: Comparision between the human and mammalian oestrus cycle smears by the main features of the cells present.
Present in high number Present in small number
From table 1, it can be seen that leukocytes are the mainly cells in di-oestrus smear. Some cornified epithelium cells are present too, and mucus fluid is detected. The number of nucleated cells appearance increases during the met-oestrus and pro-oestrus phases, as well as intermediate and parabasal cells. Leukocytes completely disappear during these two phases. In full oestrus, large non-nucleated (cornified) cells are seen with an occasional parabasal cells. Mucification of cells is a marker of the complete oestrus. If these phases are compared to the human cell cycle, it can be noted that there is a little difference in the major cell type in each phase and their features. Superficial and intermediate cells are the major cells during the whole cell cycle. Parabasal cells are present in small numbers normally. They only increase during the women's Menopausal event. Leukocytes are only seen during the severe phase, and disappear again in the premenstrual phase.
"During the anestrus portion of the cycle, the smear consists
preponderantly of leucocytes, with an occasional cornified epithelial
cell. The first appearance of estrus is marked by mucification of
the vagina, followed quickly by the complete, or nearly complete,
disappearance of leucocytes and their replacement by large numbers
of round, nucleated, epithelial cells. These cells are about
three times as large as the leucocytes and, once seen, cannot be
mistaken. At this time and for about 12 hours afterward, the
animals will breed, but at no other time. Desquamation of the
epithelial cells now occurs, and white, cheesy masses of
disintegrating squamous cells are found in the smear. Leucocytes
now again make their appearance, and the cycle is repeated."
FE D'Amour, FR Blood, and DA Belden, Jr. Manual for Laboratory
Work in Mammalian Physiology, 3rd Ed, revised, U Chicago Press,