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Cervical cancer

Cervical cancer

Figure 1. Picture provided by Roy Stewart - Cervical cancer lecture presentation.

The area joining the cervix and the vagina is the main place where cancer can form. 

The vagina has a slightly acidic environment, and this can be very harmful to the cells found on the ectocervix (also referred to as the exocervix), during puberty the cervix opens, thus in order to deal with the acidity, the cells must undergo metaplasia (genetic reprogramming of the basal cells), causing a migration of the squamocolumnar junction into the transformation zone. As a woman ages, the squamocolumnar junction becomes restored to its original site. The squamocolumnar junction and the transition zone is where most things occur, in a smear, it is required to have endocerivcal and ectocervical cervical cells, along with parts of the transformation zone.  

Cervix histology

The cervix can be divided into 2 parts:

1. Ectocervix: visible to the naked eye on a vaginal examination, it is covered by a stratified non keratinising squamous epithelium continuous with the vaginal vault. The squamous epithelium converges centrally at a small opening termed the "external os". In a woman who hasn't completed a pregnancy beyond 20 weeks (deemed as nulliparous), the os is virtually closed. 
2. Endocervix; just anterior to the os, it is lined by simple columnar, mucus secreting epithelium that dips down into the underlying stroma to produce crypts (endocervical glands).

The point of these two areas meeting is referred to as the squamocolumnar junction, its actual position can vary due to the cervical anatomy and the basal cell and subcolumnar reserve cell distribution that occurs. The progressive differentiation of these basal/reserve cells which governs the microanatomy of this region, thus resulting in migration of the SJC. The area of columnar epithelium which is replaced by squamous epithelium is referred to as the "transformation zone", it's within here and the SJC where precancerous lesions and squamous carcinomas develop.  

Human Papillomavirus (HPV)

HPV is a member of the Papoviridae virus family, and there are over 100 types of HPB which all have the ability to affect different epithelial surfaces, these include the hands and feet, as well the genital region. Some HPV types are the cause of warts and also genital warts (condyloma acuminata), HPV types 5 and 8 are associated with the development of "tree man syndrome", whereas some are the causative agent in cervical cancer. HPV has also been associated with other types of cancer, such as oral, laryngeal, anal and skin cancer in patients whom are immunocompromised. HPV types such as type 16 and 18 associated with cancer are referred to as "high-risk" HPV types, whereas those associated with benign warts, such as HPV types 6 and 11 are referred to as "low-risk" HPV types. The HPV vaccine given out to young girls in recent years targets the HPV type 16 and 18. 

Figure 2. Picture provided by Roy Stewart - cervical cancer lecture presentation

The viral capsid (fig 2) consists of 72 capsomeres, each of which is composed of 2 viral proteins, L1 (major capsid protein) and L2 (minor capsid protein). HPV uses a circle of double stranded DNA for a genome, which is around 8000 bp in length. All of the proteins the virus uses are encoded for within one strand of the DNA, thus are all read in the same direction. The genes within the DNA can be categorised into late and early, affiliated with when they're expressed during the course of an infection. 

The virus infects the basal cells of the epithelium through minor abrasions in the skin, these basal cells then divide, and their progeny will also contain HPV DNA. During the early stages of the initial infection, the number of viral genome copies is small, around 50-100, and the genome exists as an extrachromosomal DNA circle, also referred to as an episome, which goes on to replicate in the same manner as the host cell chromosomes.

The viral genome doesn't undergo amplification until the basal cells begin the differentiation process. As the cells approach terminal differentiation, the late genes L1 and and L2 are activated, and transcribe the major and minor viral capsid proteins. At this point, the viral genome number will have increased rapidly, so that each cell will produce 1000s of viral particles. As the cells approach the surface of the skin, they're shedded, and the virus particles are released in order to infect other cells and spread to other hosts. 

When the virus integrates into the hosts DNA, this is when a malignancy can arise. The plasmid linearises by losing E2 and E4, these are involved in controlling the virus and are no longer required once inserted into host DNA. With the loss of these control genes, the others become switched on. The main problem genes associated with malignancies are E6 and E7, and their regulation will become heightened. 

Figure 6.Picture obtained from Roy Stewart - Cervical cancer lecture presentation

Low grade: as cells grow and mature, they make new viruses, however as it is an episome, it doesn’t switch on the E6 and E7 genes, however there is a partial turn over of E6 and E7.

High grade: E6 and E7 switched on with no control, they are produced in a ridiculous number.

Figure 7. Picture obtained from Roy Stewart - Cervical cancer lecture presentation

It is thought that as an average estimate, cervical cancer takes around 10-15 years to develop fully. A main point is that a persistent infection is required in order for the development of neoplastic change to occur. Other cofactors are involved with the progress of the disease such as smoking, oral contraceptions etc. 

Cervical intraepithelial neoplasia (CIN)

A persistent infection can be categorised as CIN 1 (mild dysplasia), this infection can be cleared easily, however if left, it may progress into CIN 2 (moderate dysplasia) or CIN 3 (severe/invasive dysplasia), and alongside other cofactors the viral DNA will integrate itself and this can progress into cervical cancer. A screening will be able to pick up any preneoplastic changes, hence their importance.

Figure 8. Picture obtained from Roy Stewart - Cervical cancer lecture presentation

HPV binding and cellular entry

The viral particle interacts with the cellular surface it is trying to infection, through interaction between the L1, major capsid protein, and heparan sulfate proteoglycans. There is evidence which suggests that a secondary receptor is involved, possibly providing a role for the L2, minor capsid protein in interactions on cellular surface. HPV entry in intro is carried out through the binding to a cell surface receptor, whereas in vivo the basement membrane is identified as the virus binding primary site. This binding (in both scenarios) promotes a conformational change, affecting both L1 and L2 proteins, enabling them to interact with an uptake receptor. Most HPV types enter the cell via an endocytic mechanism which is clathrin-dependent. Clathrin is a protein which is associated with coated vesicle formation.  This process occurs slowly and also not at the same time, it can be associated with a long residence on the cell surface. Many details of this process however are yet to be clarified.

Host DNA integration

When the virus integrates into the hosts DNA, this is when a malignancy can arise. The plasmid linearises by losing E2 and E4, these are involved in controlling the virus and are no longer required once inserted into host DNA. With the loss of these control genes, the others become switched on. The main problem genes associated with malignancies are E6 and E7, and their regulation will become heightened. 

Malignancy

Figure 3. Picture obtained from Roy Stewart - Cervical cancer lecture presentation

Within different stages of the cell cycle, different cyclins are made, and they become activated when in the presence of their specific cyclin dependant kinase (CDK). The retinoblastoma protein (Rb), a tumour suppressor family protein, is also has a crucial role in controlling of the cell cycle. It tightly binds to transcription factors of the E2F family, and represses the transcription of particular cell cycle genes.

In the G1 phase, cyclin D becomes activated by CDK4,6, which has the ability to phosphorylate Rb, causing it to become inactivated. Cyclin E is released and activated by CDK 2, this then phosphorylates the Rb further, causing it to become hyperphosphorylated. 

Figure 4. Picture obtained from Roy Stewart - Cervical cancer lecture presentation

E7 protein  latches onto Rb via the CR2 domain, accelerating the cell into a premature state of S phase by disrupting its binding to the E2F molecule, E7 has a higher affinity for Rb than E2F thus causing its displacement. This oncoprotein is involved in both differentiation and proliferation, suggesting it can uncouple these processes  through transcription of different gene subsets. This causes eventual degradation of Rb, and as a consequence a lack of regulation within the cell cycle, promoting cellular proliferation and growth, these are hallmarks of cancer. 

E7 also affects cyclin E by deregulating its transcriptional levels, this is caused by a decrease in repression. E7 also interferes with the TGF beta tumour suppressor pathway (Smad 2,3,4), inhibiting its function.

Cyclin A alongside CDK2 becomes assembled during the S phase, and will remain active though the entirety of G2 phase also.

Figure 5. Picture obtained from Roy Stewart - Cervical cancer lecture presentation

The p53 protein is a tumour suppressor protein which regulates the cell cycle between the G2 stage and M stage of the cell cycle (the G2/M checkpoint), and is tightly regulated through MDM2. Usually, if there is an error in the DNA of a cell, the p53 protein will inhibit it's proliferation and cause the cell to carry out apoptosis. However in the presence of E6, this does not occur. E6 binds to a ubiquitin ligase called E6AP, when this E6-E6AP complex comes into contact with an p53 protein, it interacts with it and causes it to become ubiquinated. Once this happens, the p53 protein is then sent for destruction via the proteosome pathway. As the protein is degraded, it's function cannot be carried out, thus allowing damaged cells to continue through the cellular cycle and proliferate in an unregulated manner. 

E6 has another role in the up regulation of hTERT, alongside c-myc, which generates telomerase. If a loss of telomeres occurs, and a significant amount of replication occurs, the chromosomes may "stick together", if these chromosomes are then replicated again, the chromosomes may fragment. When a cell detects a shortage in telomeres, it goes into a coma like state, a cancer prevention mechanism, referred to as senescence. Germ cells do not get a shortage of telomeres, this is because their telomere mechanism is always switched on, so as to not have any anomalies in sex cells. The production of telomerase essentially expands the life cycle of a cell, thus the up regulation will cause the cell to become "immortal".  

P16 is another tumour suppressor, it's role in the cell cycle is to inhibit cyclin-dependant kinases (mostly CDK2 and CDK4) and thus halting the cell from progressing from the G1 phase to the S phase. Its production is promoted by Rb and E2F being tightly linked. However in the presence on E7, which displaces E2F, this creates an accumulation of E2F and thus an over expression of inactive P16, the high quantity of P16 can be used as a marker for the diagnosis of cervical cancer.

HPV types and diseases

There are over 100 types of HPV types, not all cause any symptoms in humans, here is a list of the most common symptoms associated with HPV and the type which causes said disease:

• Common warts: 2 and 7
• Plantar warts: 1, 2 and 4
• Flat cutaneous warts: 3 and 10
• Angogenital warts: 6, 11, 42, 43, 44, 55 and more
• Genital malignancies: 16, 18, 31, 33, 35, 39, 45, 51
• Epidermodysplasia verruciforms: 5 and 8 especially, however there are 15+ types.
• Focal epithelial hyperplasia (oral): 13 and 32
• Oral papillomas: 6, 7, 11, 16 and 32

HPV genes and their roles

• Control region: LCR
• Promoter: p97
• Oncogenes: E5, E6 and E7
• Viral replication: E1
• Viral transcription factor: E2
• Capsid formation: L1 and L2
• Viral maturation: E4

Location prevalence

Areas in which HIV are quite prominent, cervical cancer is also. This is because HIV causes those infected to become immunocompromised, thus they are significantly more susceptible to the HPV virus. Also, western countries have more modernised screening programmes, and also the use of contraception, such as condoms, is more widely used, thus incidence would be decreased. 

Risk factors

These include:

• Adolescence/young age
• Early onset of sexual activity (at, or before the age of 17, hence the HPV vaccine given to young girls)
• Number of sexual partners (e.g greater than 5)
• Risk of partners (they could be very sexually active and be of a "high" risk)
• Oral contraceptives
• Cigarette smoking (cotinine, a chemical found in tobacco, can be found within the cervical mucus, and has the ability to damage DNA)
• Lack of screening (those who've had pap smears every 3 years have 1/10 the risk of someone who hasn't had any)
• Immunosuppression

Likelihood

An approximate of 80% of the sexually active population will be involved with the HPV infection. It is very likely most people will come into contact with HPV at some point in their lives. 3/4 Americans between the ages of 15-50 have been infected by HPV at some point, however, the immune system can usually clear it. It is the most common sexually transmitted diseases. 

HPV Prevalence 

HPV can be present in a woman's cervix and vulva, inside the surrounding areas of the vagina and anus. It is common to be asymptomless. It can be obtained and then cleared without the recipient ever knowing. It is most commonly passed on unintentionally from those without symptoms, hence why it is so common. 

HPV persistence

Most women who are aware of having contracted HPV clear it, however it is unknown how many clear it completely, as it can go into remission after an active period, this could be for months or even years. Sometimes it is not entirely eliminated from the body. Infections can re-emerge from latency when said person becomes in a state of temporarily or permanent immunodeficiency  related to sunburn, pregnancy, stress both physical and emotional, cancer chemotherapy and HIV infection. It is impossible to state how long a person has been infected or when it will emerge. 

HPV and males

Men express HPV infection in the form of penile, perennial and urethral papillomas. They are much more likely to become a HPV carrier, and they will not develop cancer, as it is rare for papillomas to manifest themselves onto oncogenic cells. In order to swab for a pap analysis, a urethral swab can be taken to collect exfoliated cells however this is seldom practised. There is no FDA approved test for men thus clinically it is hard to be tested.Plus, unless there are symptoms such as aPl wart, from non-oncogenic HPV types such as type 6 and 11, men are less likely to attend any screenings.

Papanicolaou (PAP) staining

This is a classic stain, it involves the following:

• Haematoxylin stain: this is used to stain cellular nuclei, a blue - black colour.
• OG-6 counterstain: OG = Orange 6 and 6 refers to the conc. used of phosphotungstic acid, may have other variants 5 and 8. This stain colours keratin yellow.
• Second EA counterstain: EA = Eosin Azure

If performed appropriately, the stained specimen will show hues of the whole colour spectrum, red, orange, yellow, green, blue and violet. If the specimen was well prepared before the stain, the cellular nuclei will be a strong blue - black colour. Cells which have a high keratin content will be stained yellow, glycogen will also stain yellow. Superficial cells will be a orange - pink colour, and intermediate and parabasal cells will be a green/blue turquoise colour. Metaplastic cells will stain both green and pink. 

Under the microscope, it will be possible to establish whether the cells are CIN 1, 2 or 3. As the grade of the lesion increases (1-3) the cytoplasm to nucleus ratio will decrease, this is because the cells are becoming less differentiated.

Liquid based cytology

The instrument is inserted into the cervix and rotated 360 degrees in order to get cell samples from all parts, endocervix, transformation zone and the ectocervix, it is then taken out and placed into a fluid, which is then centrifuged and examined. It is them smeared out and a pap stain is used upon the sample, this provides a clean and clearer appearance. The material may last for up to 2 months and many more slides can be created from it (20-30). An automatic robot can screen these samples. Other smear tests pose problems with brushes which can damage the cells, and also with cells being left on the brushes. 

Koilocytes

These are cells which have a halo around their nuclei, this can be witnessed with a PAP smear. These cells are squamous epithelial cells which have undergone a transformation due to the infection of HPV, thus they are a positive indictor of the presence of HPV.

Smear results

If the results of a smear return back as "normal" this means there were no nuclear abnormalities  and the patient will be placed on a routine recall. 

If the results of a smear return back as "inadequate" this means there was insufficient material present, or it was poorly spread/fixed. The vision of the cells could be obscured by debris. This occurs in approximately 9% of all smears. The patient must repeat the smear, if this occurs more than 3 times consecutively, the patient must be referred for a colposcopy.

If the results of a smear return back as "border line dyskaryosis" this means that there was nuclear changes present which are not normal, however it cannot be certain whether the changes definitely represent dyskaryosis. This occurs in 5-10% of all smears. This means that the patient should repeat the smear in 6 months, as in a general case most smears will have reverted back to norma. After 3 consecutive normal smears, the patient is placed back on normal recall. If the abnormality persists after 3 smears, or gets worse, the patient is referred to a colposcopy. If in a 10 year period there are 3 borderline or results become more severe, the patient should be referred to a colposcopy. 

If the results of a smear return back as "mild dyskaryosis" this means that nuclear abnormalities are indicative of low grade CIN. The patient must then repeat the smear in 6 months. If after 3 more, results return back to normal, the patient is placed on normal recall. If the changes persist on 2 occasions  the patient is referred for a colposcopy. If in a 10 year period patient has 3 moderate results, they're referred to a colposcopy. If CIN1 is histologically confirmed, the management options are not entirely clear, depending on the results it could be watched or it could be treated.

If the results of a smear return back as "moderate dyskaryosis' this means that nuclear abnormalities reflect a probable case of CIN2, this occurs in approximately 1% of all smears. The patient will be referred to a colposcopy. If the CIN stage is histologically confirmed, treatment is provided to remove areas of abnormal cells. 

If the results of a smear return back as "severe dyskaryosis" or worse, this means that nuclear abnormalities are reflecting probable CIN3. This occurs in approximately 0.6% of smears. The patient should be referred to a colposcopy or in some rare cases, make an urgent referral to a gynaecological oncologist if invasive carcinoma is suspected. If the CIN3 stage is histologically confirmed, treatment is provided to remove areas of abnormal cells. 

NHS screening

The Nation Health Service Cervical Screening Programme (NHSCSP) is regarded as one of the best in the world, it has around 80% attendance and carries out 4 million smears a year. As a result, the number of deaths from cervical cancer in the UK and Wales is below 1000 every year. Statically due to an increase in sexual activity and a decrease in age, there should be around 5000 deaths. They offer smear tests at the age of 25 and every 3 years after that until 50 years, and from then on every 5 years. Annual tests are performed for 2 years after CIN 1 treatment, and 10 years after CIN 2 or 3 treatment. 

VILI

Squamous epithelium contains glycogen, however precancerous lesions and invasive cancer contains little to no glycogen. Iodine is glycophilic and is taken up by the squamous epithelium, staining it a mahogany brown/black colour (VILI negative). Columnar epithelium does not contain glycogen. Immature metaplasia and inflammatory lesions are at most, only partially glycogenated and when stained, it appears as scattered, ill-defined take up areas. Precancerous lesions and invasive cancer do not take up iodine, and will appear as well-defined thick mustard/saffron yellow areas (VILI positive). 

Cervical biopsies

There are two ways of sampling the the cervix, via the use of a punch biopsy, where the instrument takes small areas leaving small holes around the cervix, or a cone biopsy, where a scalpel cuts out the middle area of the cervix. 

Staging

These stages are provided by histological and clinical assessment e.g. MRI scan.

Prognosis

In accordance to the stages, here is the % survival rate of patients:

• Stage I: 91.5%
• Stage IIa: 83.5%
• Stage IIb: 66.5%
• Stage IIIa: 45%
• Stage IIIb: 36%
• Stage IV: 14%

Treatment

Vaccinations

Prophylactic vaccines are virus like particles, they consist of just the recombinant L1 structural protein, which has the ability of self assembly it contains no DNA, but the capsid itself can initiate an immune response, as it resembles intact viruses. They're non-infectious,  and very immunogenic. 

If women are vaccinated against HPB types 16, 18, 31, 33, and 45 before exposure, there will be an 85% reduction in cancer risk, decrease of 45-70% in the frequency of abnormal pap smears, a decline in abnormal pap smears within months, and a reduction in the incidence of cervical cancer would take a decade. 

However, it is important to remember, that not all cervical cancers are caused by HPV!