Cancer is a general term for over 200 diseases, used to indicate the presence of malignant neoplasms (abnormal mass of tissue). These can be characterised by the rapid and uncontrollable division of cells forming malignant tumours, whom can invade and destroy healthy tissues, and whom also persist in the same manner even when the primary stimulus causing the abnormal growth is removed. These cells have the ability to spread via invasion or implantation.
- Invasion: direct growth into tissue adjacent to the tumour.
- Implantation: growth in distant sites caused via metastasis
A tumour can be defined as any unknown lump or tissue, and can be benign of malignant.
- Benign: non-cancerous mass of localised cells which will not spread.
- Malignant: cancerous mass of cells which can spread invasively to nearby tissues (known as malignant) or to distant tissues (known as metastatic).
- Neoplasm: swelling or lump comprised of an abnormal proliferation of cells, whom are no longer under normal physiological control, of uncertain origin. The ratio of proliferation to apoptosis favours the first. Neo - new, plasma - that which is formed.
Carcinoma
Carcinoma is essentially another name for cancer, referring to a malignant tumour of epithelial origin.
Carcinoma is essentially another name for cancer, referring to a malignant tumour of epithelial origin.
This would be a tumour located in the epithelial cell layers which cover body surfaces and line the organs. Carcinomas approximately make up 80% of all cancers. Common examples would be:
- Skin
- Gut
- Breasts
- Prostate
Cancer has six main hallmarks, these are:
- Avoidance of apoptosis
- Self-sufficiency of growth signals
- Insensitivity to anti-growth signals
- Limitless replication potential
- Tissue invasion and metastasis
- Sustained angiogenesis
- Deregulation of cellular energetics
- Avoiding immune destruction
- Mutation and instability of the genome
- Tumour-promiting inflammation
Another interesting trend is the difference in the types of cancers commonly found in children in comparison to adults in 2004. In adults, 54% of cancers were derived of breast, lung, bowel and prostate cancers, whereas in children, 56% of cancers were derived of leukaemias, lymphomas and CNS/brain cancers. These findings, in my opinion, show that life style and age have a severe determining factor with adult cancers. Lung cancer is a good example of this, making up 13% of adult cancers, this will be due to the presence of smoking within the population, and the abundance of asbestos which would have been inhaled in the past couple of decades.
Cancer stages
Diagnosis of the tumour will then allow for it's prognosis, allowing to predict the tumours future activity, such as:
A benign tumour tends to be well circumscribed, can be encapsulated, is slow growing, resembles the tissue of origin and will push other tissues aside, in comparison to a malignant tumour, where there is infiltrative growth, no capsule, and will cause destruction of other tissues.
Malignant tumours
Those derived from epithelial cells, from any of the 3 germ layers, are referred to as carcinomas. A carcinoma with a glandular growth pattern would be referred to as an adenocarcinoma. Carcinomas producing squamous cells are referred to as squamous cell carcinoma. It's also general practice to mention the organ of origin, e.g renal cell adenocarcinoma. Those composed of undifferentiated unknown tissue origin, are referred to as poorly differentiated or undifferentiated malignant tumour.
Many tumours are a result of chromosomal translocations, this is an abnormal rearrangement between non homologous chromosomes, where a part of one detaches, and reattaches to another, examples of these are lymphomas, such as follicular, mantle cell, Burkitt's, and MALT.
Soft tissue sarcomas
Typically rare, only an estimated 1,300 people diagnosed per year in the UK. Occur in muscle, fat, blood vessels or any organ that supports, protects and surrounds the organs. Soft tissues of the body are referred to as mesenchyma tissues, thus sarcomas being referred to mesenchymal tumours. Generally these occur in those over 30, however some do occur in young persons. Almost half occur in the limbs, mainly near the knee joint. The chest, abdomen and pelvis are common areas. Less common are the head and neck.
Blue small round cell tumours
Small blue round cells are typically found in children and young adults with cancer, the cancers are similar in their histological features, however their diagnosis is essential as their prognosis and treatment can differ greatly. The use of karyotyping (establishing the number of chromosomes and their appearance) will provide very specific information, however in order to do this, cells must be cultured. The use of FISH would be useful.
Karyotyping
Cells which have been grown within a culture are arrested during metaphase using colchicine, they'e then placed in hypotonic solution to make them swell, and are then fixed in methanol-acetic acid. Once fixed they're placed onto slides to create metaphase spreads, and their chromosomes can be stained to create the characteristic coloured bands. An example of such a stain is "G banding", this works by the use of trypsin partially digesting histone proteins, allowing the chromosome to relax and the dye to attach itself to the exposed DNA, in this case dense A&T regions. The size and location of these bands allow cytogenetics to compare and distinguish homologous chromosome pairs. This process is extremely specific but is not cost or time effective.
In Situ Hybridisation
This is a method of localising mRNA and DNA within a cell by the hybridisation of the sequence of interest via the use of a complementary nucleotide probe. This is a morphological technique and does not cause destruction of the tissue. It's detection level is 10-20 copies per cell. The sequence of interest can range in size, from 20bp long up to 1000 bp long. It's useful for untranslated or secreted proteins. It avoids the problems of anything with shared epitopes as it's very specific. There are 5 types of probes:
Originally probes were labelled with biotin, however it has recently been discovered that endogenous biotin exists within some tissues, thus a different label is now used, such as digoxigenin (Dig) or fluorescein. The Abs for these labels are made with reporter systems attached, such as alkaline phosphatase. The advent of fluorescent labels has allowed the visualisation of multiple probes simultaneously, and resulted in the development of the FISH technique. Once only applied to metaphase chromosome spreads, it can now be used on interphase cells (interphase cytogenetics).
Benign neoplasm features
Benign tumours have specific characteristics, they:
Malignant tumours have specific characteristics, they:
Pleomorphism: variation of size and shape of cells located within a neoplasm. Mitoses on their own are not a sign of malignancy, however the presence of abnormal mitoses are, and these alongside the presence of pleomorphism and hyperchromatism (development of excess chromatin) definitely favours malignancy.
Ki 67 is a protein associated with cellular proliferation. It is found only in cells which are carrying out the cell cycle, and is brought to the surface during mitosis, however in resting cells it is not present.
Carcinoma of unknown origin
Some diagnostic techniques for malignancies can fail to determine the primary origin site where the cancer developed, if the cancer has carried out metastasis. This is referred to as carcinoma of unknown primary origin (CUPO) or occult primary malignancy. In 2007, 32,100 people (roughly 50:50 gender ratio) were diagnosed with unspecified primary cancers. In post mortem examination, 15-25% still could not be identified. This is a problem as the primary site of the cancer in most cases decides the treatment and prognosis.
The majority of CUPO are adenocarcinomas or undifferentiated tumours,however it is possible they are squamous cell carcinomas, melanomas, sarcomas or neuroendocrine tumours.
Cellular differentiation
Tumours can be "graded" on how closely they imitate parent tissues which they originate from:
TNM
This is a system for determining the anatomical extent of a disease based on the assessment of 3 components:
Both grading and staging have prognostic value, but the staging is the most valuable as it indicates extent of the disease.
Prostate
Function
Epithelia, tissue and growth disorders
When the above markers are switched on, the epithelial marks seen above in green are turned off, and the mesenchymal markers seen above in red, are turned on. Zeb 1 and 2 should also be in the list, as they are recently discovered to be down regulators of E-cadherin. These can technically be considered as growth factors as they trigger a signalling mechanism.
- Transformation stage: Single normal cell mutates into a single tumour cell
- Progression: Tumour cell replicates, and cellular variants begin to emerge, after 30 "doublings" the mass is around 1 gram in weight and contains 10^9 amount of cells. This is also known as the smallest clinically detectable mass. These cells are now mutating and becoming genetically unstable, forming different cancer cell types. Small metastases may be found in other organs. After another 10 doublings, the tumour has increased in weight to 1 kilogram, consisting of 10^12 cells, this size is considered the maximum size compatible with life. At this stage the varying cancer cells with have caused heterogeneity of the tumour. Large metastases will be present in other organs.
- Non-antigenic
- Invasive
- Metastatic
- Some will require fewer growth factors to carry out mitosis.
Neoplasia
Growth of a cancer. Size is usually bearing on no biologic behaviour. It can have varying growth configurations, such as:
Growth of a cancer. Size is usually bearing on no biologic behaviour. It can have varying growth configurations, such as:
- Infiltration and projection onto the surface
- Infiltration and ulceration of the surface
- Infiltration forming tumour mass
- Papillary
- Cauliflower appearance
- Nodular
- Lobular
- Cystic
Varying amounts of proliferating neoplastic cells and supportive stroma give tumours their excessive features.
Diagnosis of the tumour will then allow for it's prognosis, allowing to predict the tumours future activity, such as:
- Mode of spread
- Therapy response
- Genetic implications
Benign tumours
As a general rule, if -oma is a suffix then the tumour is benign, however there are exceptions, such as lymphoma, carcinoma.
The prefix coincides with the growth type, e.g:
- Adenoma (glandular)
- Papilloma (finger like)
- Cystadenoma (Cystic)
- Polp (Mucosal projection)
- Combined features such as papillary cystadenoma
A benign tumour tends to be well circumscribed, can be encapsulated, is slow growing, resembles the tissue of origin and will push other tissues aside, in comparison to a malignant tumour, where there is infiltrative growth, no capsule, and will cause destruction of other tissues.
Malignant tumours
Those derived from epithelial cells, from any of the 3 germ layers, are referred to as carcinomas. A carcinoma with a glandular growth pattern would be referred to as an adenocarcinoma. Carcinomas producing squamous cells are referred to as squamous cell carcinoma. It's also general practice to mention the organ of origin, e.g renal cell adenocarcinoma. Those composed of undifferentiated unknown tissue origin, are referred to as poorly differentiated or undifferentiated malignant tumour.
Many tumours are a result of chromosomal translocations, this is an abnormal rearrangement between non homologous chromosomes, where a part of one detaches, and reattaches to another, examples of these are lymphomas, such as follicular, mantle cell, Burkitt's, and MALT.
Soft tissue sarcomas
Typically rare, only an estimated 1,300 people diagnosed per year in the UK. Occur in muscle, fat, blood vessels or any organ that supports, protects and surrounds the organs. Soft tissues of the body are referred to as mesenchyma tissues, thus sarcomas being referred to mesenchymal tumours. Generally these occur in those over 30, however some do occur in young persons. Almost half occur in the limbs, mainly near the knee joint. The chest, abdomen and pelvis are common areas. Less common are the head and neck.
Blue small round cell tumours
Small blue round cells are typically found in children and young adults with cancer, the cancers are similar in their histological features, however their diagnosis is essential as their prognosis and treatment can differ greatly. The use of karyotyping (establishing the number of chromosomes and their appearance) will provide very specific information, however in order to do this, cells must be cultured. The use of FISH would be useful.
Karyotyping
Cells which have been grown within a culture are arrested during metaphase using colchicine, they'e then placed in hypotonic solution to make them swell, and are then fixed in methanol-acetic acid. Once fixed they're placed onto slides to create metaphase spreads, and their chromosomes can be stained to create the characteristic coloured bands. An example of such a stain is "G banding", this works by the use of trypsin partially digesting histone proteins, allowing the chromosome to relax and the dye to attach itself to the exposed DNA, in this case dense A&T regions. The size and location of these bands allow cytogenetics to compare and distinguish homologous chromosome pairs. This process is extremely specific but is not cost or time effective.
In Situ Hybridisation
This is a method of localising mRNA and DNA within a cell by the hybridisation of the sequence of interest via the use of a complementary nucleotide probe. This is a morphological technique and does not cause destruction of the tissue. It's detection level is 10-20 copies per cell. The sequence of interest can range in size, from 20bp long up to 1000 bp long. It's useful for untranslated or secreted proteins. It avoids the problems of anything with shared epitopes as it's very specific. There are 5 types of probes:
- Double stranded DNA: can be produced within bacteria via the use of plasmids, or through PCR, it has to be denatured to allow hybridisation with DNA or mRNA. They are possible to be produced in bulk, however when done so there is a risk of them re-annealing to themselves. If they're large, they may have problems when attempting to access the cell.
- Single stranded DNA: around 200-500bp in length. Can be produced via the process of reverse transcription of mRNA, or via asymmetric PCR using one primer.
- Riboprobes: these have an advantage as RNA-RNA hybrids are very strong, and are also resistant to digestion by RNases, thus post hybridisation washing with RNase can remove non-hybridised RNA and give a very clear background. These probes are hard to produce by in vitro transcription of a linearised plasmid, with an RNA polymerase from a promoter site which can produce either sense or antisense probes.
- Oligonucleotides: produced synthetically as in PCR primers, usually around 20-40bp in length. Their size is ideal for penetrating cells, however they do cover less of the target, so they're usually mixed into a cocktail in order to increase sensitivity.
- PNA probes: mimics DNA, however it has a repeating backbone of N-(2 aminomethyl)-glycine unites linked by amide bonds, The bases are attached to the backbone via methylene carbonyl linkage. It cannot be used as a PCR primer.
Originally probes were labelled with biotin, however it has recently been discovered that endogenous biotin exists within some tissues, thus a different label is now used, such as digoxigenin (Dig) or fluorescein. The Abs for these labels are made with reporter systems attached, such as alkaline phosphatase. The advent of fluorescent labels has allowed the visualisation of multiple probes simultaneously, and resulted in the development of the FISH technique. Once only applied to metaphase chromosome spreads, it can now be used on interphase cells (interphase cytogenetics).
Benign neoplasm features
Benign tumours have specific characteristics, they:
- Grow slowly
- Are encapsulated within a fibrous capsule
- Have a smooth surface
- Compress surrounding organs and tissues, pushes them to the side
- Small in size (usually)
- Do not cause death unless occurring in the central nervous system, or compressing vital organs
- Cells are very differentiated, thus resemble the tissue of origin
- Cells are uniform and almost identical
- Blood vessels around the tumour are well formed
- Doesn't have necrosis
- Doesn't metastasise
- DNA content is normal
- Karyotype is normal
Malignant tumours have specific characteristics, they:
- Grow rapidly
- Do not have a capsule
- Have an irregular surface
- Tumour invades and destroys surrounding tissue
- Large in size (usually)
- Causes death if not treated
- Cells are not very well differentiated, especially in comparison to benign tumours
- Malignant neoplasms mostly do not resemble origin tissue
- Cellular nuclei can be enlarged and hyperchromatic (darker nucleus)
- Numerous prominent nucleoli
- Cellular pleomorphism
- Increased mitotic activity with abnormal mitotic figure
- Blood vessels surrounding the neoplasm are numerous and poorly formed, some do not have any endothelial lining, and this can leads to tissue haemorrhaging
- Necrosis is present
- Metastasis does occur
- DNA content of the cells tends to be increased, additional chromosomes can be present
- Karyotyping abnormalities such as aneuploidy and polyploidy occur.
Pleomorphism: variation of size and shape of cells located within a neoplasm. Mitoses on their own are not a sign of malignancy, however the presence of abnormal mitoses are, and these alongside the presence of pleomorphism and hyperchromatism (development of excess chromatin) definitely favours malignancy.
Ki 67 is a protein associated with cellular proliferation. It is found only in cells which are carrying out the cell cycle, and is brought to the surface during mitosis, however in resting cells it is not present.
Carcinoma of unknown origin
Some diagnostic techniques for malignancies can fail to determine the primary origin site where the cancer developed, if the cancer has carried out metastasis. This is referred to as carcinoma of unknown primary origin (CUPO) or occult primary malignancy. In 2007, 32,100 people (roughly 50:50 gender ratio) were diagnosed with unspecified primary cancers. In post mortem examination, 15-25% still could not be identified. This is a problem as the primary site of the cancer in most cases decides the treatment and prognosis.
The majority of CUPO are adenocarcinomas or undifferentiated tumours,however it is possible they are squamous cell carcinomas, melanomas, sarcomas or neuroendocrine tumours.
Cellular differentiation
Tumours can be "graded" on how closely they imitate parent tissues which they originate from:
- Well-differentiated: the cells are very similar in both appearance and conformation to the tissue of origin
- Poorly-differentiated: the cells only show minimal resemblance's to the normal parent tissue
- Anaplastic: tumour shows no obvious similarities to the origin tissue, this type is associated with having aggressive behaviour.
The grading is based upon the appearance of the cells under a light microscope along with H&E staining. A higher grades means a smaller degree of differentiation, thus the worse the biological behaviour. Most systems have 3 or 4 grades:
- Grade 1: Well differentiated
- Grade 2: Moderately differentiated
- Grade 3: Poorly differentiated
- Grade 4: Anaplastic
Functional differentiation
Well differentiated tumours produce the normal product of the origin tissue, however poorly differentiated tumours are less likely to carry out the standard functions. This differentiation provides clues as to the clinical aggressiveness of the tumour. Tumours can lose their differentiation features over time as they become more malignant, and acquire more genetic mutations. The level of differentiation within the cells depict how they will respond to certain therapies and treatments.
Spreading pathways
There are different pathways taken during metastasis of malignant cells:
- Lymphatic spread: usual route for carcinomas, involves the lymph nodes
- Haematogenous spread: usual route for late stage carcinomas
- Transcoelomic (body cavity) spread: seeding of cavities surfaces by tumour, typical in ovarian carcinomas
TNM
This is a system for determining the anatomical extent of a disease based on the assessment of 3 components:
- T: the extent of the primary tumour
- N: the presence or absence and extent of regional lymph node metastasis
- M: the presence of absence of distant metastasis.
The addition of numbers to these 3 components indicates the extent of the malignant disease. The system is a shorthand notation for describing the extent of a particular malignant tumour:
TX - Primary tumout cannot be assessed
T0 - No evidence of primary tumout
Tis - Carcinoma in situ
T1,2,3,4 - Increasing size and/or local extent of primary tumour
NX - Regional lymph nodes cannot be assessed
N0 - No regional lymph node metastasis
N1 - Regional lymph node metastasis
MX - Distant metastasis cannot be assessed
M0 - No distant metastasis
M1 - Distant metastasis
Both grading and staging have prognostic value, but the staging is the most valuable as it indicates extent of the disease.
Prostate
Function
The vagina has an environment which is slightly acidic, with approximately a pH of 4, this is to protect against pathogens. Sperm however, are acid-sensitive, thus their environment, semen, is alkaline. Due to this, any ejaculate abolishes the vaginas protective acidity for many hours after it's entry, this is to ensure survival of the sperm. Seminal vesicle secretions are rich in fructose, as this is a primary energy source for sperm, and also rich in prostaglandins and proteins which promote semen clotting in the vagina. These secretions are also rich in Ca2+, zinc, citric acid, acid phosphatase, albumin and prostatic specific Ag (PSA). PSA has the ability to reverse the clotting enzyme affect from seminal vesicle fluid. he enzyme has the ability to make ejaculate gel and "glue" in place in the female cervix. Sperm will remain within this gel, until the PSA enzyme dissolves the clot and allows sperm to swim into the uterus.
The exocrine gland is the size of a walnut, and weights approximately 20 grams. Within the adult prostatic parenchyma may be divided into 4 biologically and anatomically distinct zones or regions:
- Peripheral
- Central
- Transitional zones
- Region of the anterior fibromuscular stroma
These are important as different types of proliferative lesions may arise in each region. For example, most hyperplasias arise in the transitional zone, and most carcinomas originate in the peripheral zone.
This is important to mention in the exam. Try and draw it in the exams if needed (not a rude picture though!).
This is important to mention in the exam. Try and draw it in the exams if needed (not a rude picture though!).
Figure 1. Picture provided by Roy Stewarts "Prostate" lecture presentation.
Histology
This is very important to include:
It is very important that the normal histology is known, as this is the main way to diagnose benign vs malignant tumours. This is also used in the grading of prostate cancer. Essentially, the prostate is a gland composed of tubular glands which have 2 layers of cells:
It is very important that the normal histology is known, as this is the main way to diagnose benign vs malignant tumours. This is also used in the grading of prostate cancer. Essentially, the prostate is a gland composed of tubular glands which have 2 layers of cells:
- Basal cuboidal layer: rests of a basement membrane
- Inner columnar luminal layer
The glands can be rounded or have papillary type (nipple like) projections. This can be useful diagnostically. The rest of the gland is background stromal cells consisting of fibro-muscular tissue, which has an influence of the glandular tissue itself, and not just inert background material.
Normal histology and immunohistochemistry
The basal layer is positive for high molecular weight cytokeratins and the mouse antibody cocktail anti-cytokeratin 34bE12 reacts with cytokeratins 1,5,10 and 14. P63 is also positive, and it has been shown that without P63, mice do not develop a prostate, thus a cocktail of 34bE12 and P63 is a good marker, in order to pick up all of the basal cells. Remembering the names of this will give you more marks. Corpora amylacea - normally found in normal prostate.
Benign prostatic hyperplasia (BHP)
Major theories attempting to explain the aetiology of pathological phase BPH include:
- Dihydrotestosterone (DIT) hypothesis: this is a shift in prostatic androgen metabolism that occurs due to age, leading to abnormal accumulations of DHT, producing an enlarged prostate.
- Embryonic reawakening theory: a reawakening of the embryonic induction potential of prostatic stroma - a change in the prostatic stromal-epithelial interaction that occurs alongside ageing, leading to an a promoting effect on prostatic growth.
- Stem cell theory: featuring an increase in the total prostatic stem cell numbers and/or clonal expansion of stem cells into amplifying and moving cells which occurs with ageing.
- Inflammatory theory: Prostatic inflammation may contribute to the growth of the prostate, due to the inducing of cellular growth because of the presence of inflammatory markers and agents which have growth stimulating abilities.
BHP involves the enlargement of glandular and stromal tissue, stromal being mainly fibro muscular. Each can have varying amounts, providing a wide range of histological BPH presentations. The main activating component is DHT, that is converted within stromal cells from testosterone via enzyme 5alpha reductase, which is also produced by stromal cells (see fig 1). DHT has a function as an autocine/paracrine agent, causing direct cell stimulation, or up regulating mitogenic signals which act upon the proliferation of neighbouring cells. Testosterone as similar attributes to DHT, but it is nowhere near as potent. A reduction in the activity of DHT will cause the gland to shrink, however if the gland is very large, it may have to be dealt with surgically, e.g by use of trans urethral resection prostate (TURP). This can produce prostatic chippings, these must all be processed as there may be a background micro foci of cancer or PIN.
Figure 1. Picture provided by Roy Stewarts "Prostate" lecture presentation.
DHT binds to nuclear androgen receptors in both epithelial and stromal prostate cells, DHT has a higher affinity for AR than T, forming a more stable complex. DHT binding promotes the transcription of androgen-dependent genes. DHT itself doesn't promote mitosis, however the DHT-mediated transcription of genes results in the production of many growth factors, such as FGF (fibroblast growth factor), with FGF-7 (keratinocyte growth factor) being important. Others produced are FGF 1 and 2, and TGF beta, that promotes the proliferation of fibroblasts. The overall cause of BPH isn't known, however DHT related growth factors ac by increasing stromal cell proliferation, and lead to a decrease in epithelial cells apoptosis.
The main feature of BPH is the presence of nodules. Their composition range from purely stromal and fibromuscular, to fibroepithelial with a glandular predominance. Glandular proliferation takes the form of aggregations of small to large cystically dilated glands, lined by 2 layers, inner columnar and an outer cuboidal or flattened epithelium.
BPH diagnosis cannot be made using a needle biopsy, as the histology of glandular or mixed glandular-stromal nodules cannot be appreciated in limited samples. They do not necessarily sample the transition zone where BPH occurs as this is in the middle of the prostate.
5alpha reductase inhibition
Finasteride is used as an inhibitor for 5alpha reductase, it can inhibit its action by around 80-90%. As a result, the amount of DHT is reduced by 70% in serum, 90% in prostate and 34% in skin. The amount of testosterone increases by around 10%, and PSA is reduced by an average of 50%, as a result of this, the total prostate volume will decrease by 15-25%.
BPH treatment
For cases which are moderate to severe that are resistant to medical therapy, there are a range of invasive procedures which can be used instead. TURP has been good in terms of reducing symptoms, improving flow rates and decreasing post-voiding residual urine. It is now used as the first line of therapy for those under certain circumstances, such as those with reoccurring urinary retention. However, as a result of its morbidity and cost, other procedures have been developed. Such as:
- High-intensity focused ultrasound
- Laser therapy
- Hyperthermia
- Transurethral electrovaporisation
- Transurethral needle ablation using radiofrequency
- Prostate artery embolisation as recently been trailed as a therapy also
Nodular hyperplasia is not considered to be a premalignant lesion.
Prostate cancer
Prostate cancer is the most common cancer within men. Statistics show approximately every hour one man dies from prostate cancer, meaning over 10,000 every year. Over 40,000 men are diagnosed with prostate cancer every year, more than 100 a day. Estimated that in 2030, prostate will be the most common cancer type. 1 in 8 men will get this. There are many risk factors which aid in the formation of prostate cancer:
- Age
- Family history: this is one of the strongest risk factors, 5-10% of PC cases and 30-40% of early onset cases (those who develop it before the age of 55) are caused by inherited susceptibility genes. There is a 2-3x increase in risk for those who have a first degree relative diagnosed with PC, if the relative was under the age of 60, or the person has more than one relative with PC, the risk exceeds the average by 4x. If the person has more than one relative when an early onset of PC, the risk increases 7x. If there is a history of breast cancer, especially under the age of 60, this will also increase the risk, due to an association with BRCA1 and 2. Studies have identified a number of risk-associated loci, including one at 8q24 which appears to selectively increase the risk among African men.
- Ethnicity: the incidences of which PC occur differ in varying locations in the world, suggesting ethnicity is a factor. In the UK, black caribbean and african men have 2-3 x the risk of developing PC than white men, and asian men have an even lower risk, this trend is also seen in the US.
- Life style: migration studies have shown that men moving from low risk to high risk areas have showed an increase in cases, suggesting that life style factors of the area also play a role, e.g a South Asian man living in the UK has a higher chance at developing PC than those living in South Asia.
- Diet
- Alcohol and smoking
- Body weight and physical activity
- Medications, medical procedures and infections
- Endogenous hormones
- Diabetes mellitus
Cancer and gene arrangements
There is a common somatic mutation in prostate cancer which gives rise to chromosomal rearrangements (translocations) that juxtapose the coding sequence of an ETS family transcription factor gene (most commonly ERG and ETV1) next to the androgen-regulated TMPRSS2 promotor, testosterone increases. ETS over expression makes normal prostate epithelial cells more invasive, maybe by the up regulation of MMPs. Tumours with rearranged ETS genes do not necessarily have certain distinctive morphologic features, and a rarely have a differing gene expression signature than those who do not have different ETS arrangements, thus suggesting that ETS gene rearrangements define a specific molecular sub-class of prostate cancer. They may also have implications for prostate cancer during screening and early diagnosis, it's possible to detect ETS fusion genes in urine, through the use of PCR. Most aggressive fast growing PCs have these translocations.
CAG repeat
Endogenous hormones, including androgens and oestrogens, have a likely influence of the carcinogenesis of the prostate. Men who have been castrated, such as eunuchs, whom have levels of testosterone equal to that of before puberty, will not develop prostate cancer. Thus, it is possible that a change in androgen biosynthesis and metabolism e.g the role of the androgen receptor (AR) CAG repeat in exon 1, could influence the risk of PC. It has been reported that AR CAG repeat length greater than, or equal to 20 repeats, conferred a protective effect for PC in men. Long repeats make the cell less likely to divide.
Androgen resistant cancer
The term "castration-resistant prostate cancer" or CRPC arose from what used to be referred to as androgen-independant prostate cancer. The AR is considered the principle driver towards prostate cancer progression. This concept showed that castration temporarily slowed the progression of PC, however subsequent castration-resistant growth of PC has been attributed to a variety of mechanisms, including:
- activation by receptor tyrosine kinases from growth factors,
- loss of cell cycle regulators and someones
- genomic mutations in the AR allowing response to nonspecific AR ligands.
- It has been showed that increased AR expression was the most common event associated with CRPC growth.
- It has also been shown that metabolic adaptation is involved also, involving the production of androgen within the tumour cells, self stimulatory.
- Many studies have revealed there are many alternative splice forms of AR, these variants have differing structures, however each variant lacks portions of the ligand-binding domain (LBD), a feature predicted to produce a constitutively active receptor.
- The variants increased expansion was associated with a more rapid disease occurrence, following radical prostatectomy for localised disease.
Aggressive cancer "Pussycat vs Tiger"
5 single nucleotide polymorphisms (variations within a gene) were located in, or associated with 5 genes which may affect PC progression:
- LEPR: This is the strongest marker with PC mortality, it aids in tissue growth control, inflammation, development of blood vessels and the density of bones. These make this gene an interesting candidate for understanding disease progression, as the primary metastatic site for PC is bone, and these metastases are predictive of fatal disease.
- RNASEL: This gene is associated with hereditary PC, and associated with apoptosis, inflammation and the proliferation and adherence of cells (cancer growth hallmarks).
- IL4: Interleukin 4, associated with growth of tumours, blood vessel development and cancer cell migration.
- CRY1: Cytochrome 1, gene that impacts the circadian rhythm this possibly affecting androgen levels.
- ARVCF: member of the catenin protein family, aids in the communication of inside and outside cells. Increased expression has been shown to disrupt adhesion of cells, possibly facilitating cancer progression.
Patients whom carried 4 or 5 of these markers had a 50% increased risk of dying of PC than those with 2 or less. There is a correlation with death rate and the number of SNPs present.
BRCA2
BRCA2 is now not only associated with early development of PC, but also with the aggressive form. It is found in 1:100 of PCs. If there is a family history of breast or prostate cancer, then this gene should be tested for, however this isn't currently available within the UK.
Prostate specific antigen (PSA)
PSA is often used as a tool for screening PC, however elevated levels can also be present in benign tumours, and some malignancies may have low levels, however the cut off is given at 4.0 ng/ml. A better way to measure this gene is via its velocity, or rate of change over time, the value 0.75 ng/mL per year best distinguishes between men with and without PC. In order to be considered efficient, there must be a minimum of 3 tests over a period of 1.5-2 years. Best used as a marker of treatment.
PSA vs volume: including the ratio of serum PSA value and volume of prostate gland (PSA density). Men with enlarged hyperplastic prostate glands have a higher overall serum PSA than men with smaller glands. Measuring serum PSA density factors out benign prostatic tissue contribution to serum PSA levels. This is calculated by diving the total serum PSA level by the estimated gland volume (determined by transrectal ultrasound measurements) in order to estimate the PSA produced per gram of prostate tissue.
As men age, their prostates tend to become enlarged with BPH. Overall older men would have a higher serum PSA level than younger men. Thus there are reference ranges for upper age-specific PSA:
- 2.5 ng/ML - 40-49 years of age
- 3.5 ng/mL - 50-59 years of age
- 4.5 ng/mL - 60-69 years of age
- 6.5 ng/mL - 70-79 years of age
Free PSA: Immunoreactive PSA (the form which is detected by antibody tests) exists in 2 forms, a major fraction bound to alpha1-antichymotrypsin, and a minor free fraction. The % of free PSA (free PSA/total PSA x 100) is lower in men with PC than those with BPH. Free PCA higher than 25% indicates a lower risk of cancer, as compared to free PSA values of less than 10%, which can cause concern for cancer.
Biopsies
Talk about this more (will be updated soon). Braccotherapy (implant therapy) - insert a core of radioactive material.
Biopsies
Talk about this more (will be updated soon). Braccotherapy (implant therapy) - insert a core of radioactive material.
Histological diagnosis of PC
Most lesions are adenocarcinomas which produce well-defined, readily demonstrable gland patterns. These glands are typically smaller than benign glands, and are lined by a single uniform layer of cuboidal or low columnar epithelium. In contrast to benign glands, which have two layers, the outer basal cell layer found in benign glands is absent in adenocarcinomas. These inner cells cannot be stained, whereas the outer basal cells can. PC glands are crowded, and lack branching and papillary infolding. The cytoplasm of the tumour cells range from pale-clear, as noted in benign glands, to a distinctive amphophilic appearance. The nuclei in PC are large, and sometimes a cell can contain more than one. There is also variation is nuclear size and shape, however blatant pleomorphic features are not present. Mitotic figures are uncommon.
Diagnosis
Difficulty in establishing an appropriate diagnosis with PC stem, not only due to the small amount of tissue available for examination from the needle biopsy, but also that a biopsy often only samples a new malignant glands among many benign glands. Morphologically, PC malignancy clues are very subtle, again making it hard to efficiently diagnose. There are many benign mimickers of cancer which can lead a pathologist to a misdiagnosis. There are very few histologic findings on a biopsy which are specific PC, however a perineural invasion is one. The general diagnosis is made based on a combination of morphological and ancillary findings.
Diagnostic aids: A distinguishing feature between benign glands and malignant glands is the presence of basal cells, these are only present in benign glands. This can be exploited by the immunohistological use of markers which are specific to basal cells, such as high molecular weight cytokeratins 34bE12, 1, 5, 10, 14 and also P63. Another marker which is very selective for PC cells and not normal cells is AMACR (alpha-methylacyl-coenzyme A-racemase), this is up regulated in PC and can be observed by the use of immunohistochemistry. The majority of PCs are positive for AMACR, with sensitivities varying from 82%-100%. AMACR is a positive stain for malignancy, must be used with H&E.
AMACR
P504S is a 382 aa cytoplasmic protein which has been recently identified by microarray screening of prostatic carcinomas, it has been identified with AMACR, which is an enzyme that catalyses the racemization of alpha-methylacyl branches carbolic coenzyme A thioesters. It is located within the mitochondria and peroxisomes, where it aids in the oxidation of branched chain fatty acids. It is also responsible for the racemization of an intermediate in the synthesise of bile acids. In neoplasia, AMACR is most often expressed in tumours associated with a high fat diet, such as colonic and prostatic carcinoma. AMACR stains red, and CK and P63 stain brown.
Prostatic intraepithelial neoplasia (PIN)
In around 80% of cases, prostatic tissue removed from a carcinoma will also contain presuming precursor lesions, referred to as PIN. These consist of architecturally benign prostatic acini (sac like cavity) lined by cytologically atypical cells with prominent nucleoli. PIN and carcinomas may be identical cytologically, however architecturally PIN involves larger branching glands with papillary infolding, compared to invasive cancer that is typically characterised by small crowded glands with straight luminal borders. PIN glands are surrounded by a layer of basal cells, alongside an intact basement membrane - there are many types of evidence relating PIN to invasive cancer. For example, both typically predominate in the peripheral zone, and are uncommon on other zones.
Comparing a non cancerous prostate to one with cancer, those with have a higher frequency, and a greater extent of PIN. PIN is often observed near cancer, sometimes with cancer budding off from it. A high number of molecular changes observed in invasive cancers can also be witnessed in PIN, providing evidence that PIN is a type of intermediate between normal and invasive cancer. But how PIN behaves and develops into cancer is not known. thus as used in cervical cancer, the term "carcinoma in situ" cannot be applied to PIN. However, PIN is important in identifying BPH chippings.
Benign glands have a continuous basal layer, PIN glands have a fragmented basal layer, and malignant glands have no basal layer.
Grading
The Gleason system is used to grade prostate cancer. PCs are stratified into 5 grades, on the basis of their glandular patterns of differentiation. It's a subjective assessment.
- Grade 1: represents the most well-differentiated tumours, in which the neoplastic glands are uniform and round in appearance and are packed into well-circumscribed nodules.
- Grade 5: tumours show no glandular differentiation, and the tumour cells infiltrate the stroma in the form of cords, sheets and nests.
All other grades fall in between these.
Most tumours contain more than one pattern, so a grade is given to the most dominant pattern, and another grade to the most frequent pattern, the two grades are then added together to give a combined Gleason score, thus the number can be between 2 and 10. The scores are combined into groups with similar biological behaviour;
- 2-4: represent well differentiated cancer, typically found in small tumours within the transition zone. In surgical specimens, low-grade cancer is typically an incidental finding on TURP, which is performed for symptoms of BPH.This is why all chippings are sampled.
- 5-6: intermediate grade tumour: the majority of potentially treatable cancers detected on a needly biopsy as a result of screening have a score of 5-7.
- 7: moderate to poorly differentiated tumour
- 8-10: high-grade tumour, these tend to be advanced cancers that are unlikely to be cured.
Although PCs can become more aggressive with time, the score usually remains stable over several years. Grading is very important in PC as the grade and stage are the best prognostic predictors.
The state of DNA ploidy can affect the Gleason score, with the score usually being higher when DNA ploidy is abnormal.
Staging and risk
Low risk; clinical stage T1c, or T2a, PSA level less than 10 ng/ML, GS of 2-6.
- Very likely the disease is within the prostate (confined)
- Minimal risk of spread to distant sites such as bones
- Prostate-only treatment (implant alone) is appropriate
- Observation is an appropriate option in selected individuals
- Extremely high cure rate with all therapies
Intermediate risk; clinical stage T2b, PSA 10-20 ng/mL, GS of 7.
- Possible disease is beyond the prostate (not necessarily confined)
- Low but significant risk of metastasis
- Implant alone is appropriate for some patients, however combination therapy is an excellent option
- Observation is rarely appropriate
- High cure rate with implant based therapies
High risk; clinical stage T2c, PSA over 20 mg/mL, GS of 8-10
- Very likely disease has progressed beyond the prostate
- Probable risk of metastasis
- Combination therapy is needed
- Observation is never appropriate
- Curable with intensive treatments - body wide treatments such as hormone therapy are often combined with radiation treatments.
Recent studies have indicated the presence of the gammaretrovirus XMRV being present in a significant amount of PCs, this may be of relevance?
Papanicolaou (PAP) staining
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:
- 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.
- 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.
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!
Epithelia, tissue and growth disorders
Certain cells of the body are specialised for specific functions. A tissue is defined as a group of cells with a similar structure and function, there are 4 main types of tissue:
- Epithelium
- Connective tissue
- Nervous tissue
- Muscle
Epithelia are derived from 3 layers, referred to as the germ layers; the ectoderm, mesoderm and endoderm. They line and cover all of the body's surfaces with the exception of articular cartilage, tooth enamel and the anterior iris surface. Its basic functions, dependent on its location are:
- Protection: for the skin
- Absorption: for the small and large intestines
- Transport: on the surface mediated by cilia cells
- Secretion: for glands
- Excretion: for the tubules of the kidney
- Gas exchange: for lung alveolus
- Gliding: between surfaces, for the mesothelium
Once fertilisation occurs, the sperm and egg form a zygote, then grow into a blastocyst, and then become what is referred to as a gastrula. The ectoderm, the external layer of the gastrula, derives epithelia such as skin cells, brain neurones and cell pigments. The mesoderm, the middle layer of the gastrula, derives cardiac muscle, skeletal muscle, kidney cell tubules, red blood cells and smooth muscle. The endoderm, the internal layer of the gastrula, derives the lung alveolar cells, thyroid cells and pancreatic cells. Germ cells are also derived from the gastrula, these make up sperm cells in males and egg cells in females. Sometimes however these cells can end up in random places, causing disfigurements, such as teratomas. Germ cells are totipotent, meaning they can form anything derived from the germ layers.
Due to their location and functions, epithelial cells are renewed continuously by mitosis, this however makes them prone to causing cancers. They lack a direct blood or lymphatic supply, thus their nutrients have to be provided via diffusion. There is almost no free intercellular substances. The structural organisation of the epithelium is maintained by cell adhesion molecules and junctional complexes. The cells are anchored to a basal lamina, this and connective tissue components interact in oder to form the basement membrane. The cells have a structural and functional polarity, apical and basal (top and bottom), however in cancer, this polarity is lost.
Epithelia classification
This classification is based upon the cellular morphology, not the cellular functional characteristics. It is determined by their shape and arrangement.
Shape can be:
- Squamous: very flat, almost 2 dimensional squares
- Cuboidal: 3 dimensional squares, cubes
- Columnar: 3 dimensional columns, rectangular
Arrangement can be:
- Simple: single layer
- Stratified: multilayered
- Pseudo-stratified: technically a single layer, but looks double as cells are in a top and tail position
- Transitional: multilayered however can be relaxed and distended
Figure 1
Simple epithelium
Simple squamous is a single layer of flat cells, and usually forms membranes. It lines body cavities and lungs and capillaries. It is given the name endothelium when it lines the blood and lymphatic vessels, and mesothelium when lining the body cavities. Due to their thin nature, they're useful for gaseous exchange in the alveolar.
Simple cuboidal is a layer of cube like cells, they're common in glands and ducts. They also form the walls of kidney tubules, and cover the ovaries. This is a large contributor to the incidences of ovarian cancer, a theory suggests that every time an egg is released, it may rupture the outside of the ovary where it will then have to heal, thus the longer the ovulation period, an increase in the chance.
Simple columnar is a single layer of tall, rectangular cells. They often include goblet cells which secrete mucus, they also line the digestive tract.
Pseudostratified is a single layer of cells, however some are shorter than others giving it the appearance of a double layer, sometimes it can be ciliated, this occurs in the respiratory tract. They may also have a absorption or secretory function.
Stratified epithelium
Stratified cuboidal cells are multilayered, and they ave an apical surface (the top of the top layer). It is rare if surface cels are cuboidal, this only occurs in sweat gland ducts and the male urethra.
Stratified squamous cells of the free edge are flattened, they're usually found as a protective covering where friction on the area would be common, such as the skin,mouth and oesophagus. These can be keratinised also.
Transitional epithelium
Shape of the cells will depend upon the amount of stretching required, these kind of cells line the urinary system.
Microvilli
These are finger-like processes which extend from the apical surface of cells, usually around 1-2micrometres in length. Their main component is their core of actin filaments, which are around 6nm thin, these are named microfilaments. They're very extensive in any absorptive epithelium, as they increase the cells surface area, and can be referred to as a striated border or brush border. Bundles of actin filaments extend into a part of the cell which is named the terminal web (see figure 2), these are actually anchored to the cell.
Figure 2.
Cilia
Cilia are long cytoplasmic extensions around 5-10micrometres in length. These do not contain actin filaments. Instead they have a complex arrangement of microtubules (9 around the edges and 2 in the centre) named "axoneme", which have small appendages named dynein arms which allow for movement, they're essentially molecular motors, thus the main component of cilia is tubulin. Cilia facilitate flow of fluid over an epithelium, for example on the trachea, a tubular organ. The structure is the same as flagellum (see fig 3).
Figure 3.
Kartagener's Syndrome (KS)
This autosomal recessive disease is essentially immotile-cilia syndrome. It occurs in around 1 out of 32,000 live births in the US. The symptoms include chronic upper and lower respiratory tract disease, as a result of ineffective mucociliary clearance, sterility in males, as the flagella in the sperm doesn't work properly and approximately 50% of those suffering with the syndrome display situs inversus, which is where the visceral internal organs are mirrored (different sides to normal), also referred to as transposed viscera. The biopsy examinations reveal abnormal/non motile cilia. The axoneme is lacking in dynein arms.
Basement membrane
This is an extracellular component which is in direct contact with the basal domain of epithelial cells, it can be made visible under a light microscope with the use of a periodic acid-Schiff (PAS) stain, making it PAS positive. The basement membrane can be defined into 2 layers/laminae:
- Basal lamina: this contains laminin, fibronectin, type IV collagen, heparan sulfate proteoglycans and nidogen.
- Reticular lamina: contains type 3 collagen.
The main components of these 2 layers are glycoproteins. They can be witnessed under the electron microscope.
Basal lamina functions
The basal lamina as a variety of functions:
- Adhesion of epithelial cells to underlying CT, in skeletal muscle the BL maintains the integrity of the tissue, and any disruption to it will cause muscular dystrophies.
- Act as a selective permeability barrier, with the filter being based upon the charge of the molecule and its size.
- Good substrate of cellular migration, for example during wound repair or embryonic development.
- As a barrier critical to metastatic potential in epithelial cancer cells referred to as carcinomas. A carcinoma which is in situ means it has not breached the basement membrane.
The main protein making up the BL is type 4 collagen, this forms flat sheets and has the ability to self polymerise. Perlecan also has the ability to self polymerise, and laminin also has the ability to self polymerise and also can interact with itself.
Secreted protein acidic and rich in cysteine (SPARC)
This is also known as osteonectin. It is expressing during many stages of development in a variety of organisms, however it is restricted in adult vertebrates primarily to tissue which under consistent turnover or remodelling, such as the bone. The over expression of SPARC is found in various human malignancies.
Melanoma cells with suppressed expression of SPARC could no longer generate tumours, however melanoma cells which induced expression of SPARC has 100% tumorigenicity. High SPARC levels are also associated with metastatic tumours, as it induces the expression of MMPs, MMP1, MMP2 and MMP9, which are involved in the degradation of basement membranes and extra cellular matrix. This is crucial process for invasion and metastasis in human malignancies.
Metastatic cascade
Tumour cells coat themselves in the host platelets in order to avoid detection from the immune system. Collagen is an example of an angiogenesis inhibitor. Arrestin, canstatin, turnstatin and endostatin, all type 4 collagen have specific anti-angigenic properties. Without angiogenesis, a metastatic deposit will only grow around 2mm before dying. If the integrin binding it is inhibited, then so it growth of the tumour.
Cadherins
The name is derive from the term "calcium-dependent adherence protein", the family consists of around 90 members, whom al participate in the interactions between cells of the same type, which is ideal for epithelial layers. These interactions which connect the plasma membranes of adjacent cells, forming 2 cellular junction types:
- Zonula adherens: these are small, spot like junctions located near the apical surface of epithelial cells
- Desomosomes: stronger and more extensive junctions, present in epithelial and muscle cells.
Linkage of cadherins with the cytoskeleton occurs through 2 classes of catenins; beta-catenin links cadherins with alpha-catenin which in tern connects to actin, thus completing the connection with the cytoskeleton.
E cadherins
These mediate homotypic adhesions in epithelial tissue, thus serving to keep the epithelial cells together, and to relay signals between the cells. In several epithelial tumours, e.g adenocarcinomas (gland cancer) of colon and breast, E-cadherin is down regulated, this reduces the cells ability to adhere to one another and promotes their detachment from the primary tumour, and also their advance into other tissues. In some tumours, E-cadherin is normal, but its expression is reduced due to mutations for one of the catenin genes.
Epithelial-mesenchymal transition (EMT)
This is the name given to the process in which epithelial cells lose their polarity and cell to cell adhesion, and gain invasive and migratory properties, turning them into mesenchymal stem cells, a multi-potent type of cell which has the ability to differentiate into many cell types.
EMT is crucial in a number of developmental processes, including mesoderm formation, neural tube formation, and foetal development (embyrogenesis). It is also found to occur in wound healing, and in the initiation of metastasis for cancer progression. Normal cells have contact inhibition, or spatial awareness, however cancer cells do not. The cells have an amoeba shape, they detach and become invasive and motile, they break through the basement membrane and reach the blood vessels in order to move to another location. Once they have found a place to form and grow, the process is reversed by a process called mesenchymal-epithelial transition. In order to check for this, a stain can be carried out which will show that any mesenchymal cells will not contain E-cadherin.
When the above markers are switched on, the epithelial marks seen above in green are turned off, and the mesenchymal markers seen above in red, are turned on. Zeb 1 and 2 should also be in the list, as they are recently discovered to be down regulators of E-cadherin. These can technically be considered as growth factors as they trigger a signalling mechanism.
Cellular adaptation
Adaptive responses result in an increased tissue mass, the increased functional demand or endocrine stimulation are what usually cause hypertrophy (increase in volume) and hyperplasia (increase in cell number). These new patterns of growth are stable whilst the causative stimulus persists, however once it is removed, the tissue will return to normal growth patterns.
Size
Acquired changes may be physiological or pathological, this is due to external influences, however these can potentially be reversible.
Atrophy: this is a reduction in size or activity. Examples of a physiological atrophy are the thymus as it ages, a thymus in a child is much more larger and dense in comparison to a thymus in an adult, ovaries after menopause and immobilisation of a broken limb. An example of a pathological atrophy are denervation, loss of trophic hormone, reduced blood supply and excessive pressure.
Hypertrophy: this is an increase in cell size. Examples of a physiological hypertrophy are increased skeletal muscle bulk due to training, or the uterus during pregnancy. A pathological example of hypertrophy is cardiac muscle due to an abnormal increase in workload, e.g. aortic valve stenosis. Mechanism sensors appear to be the major triggers for physiological hypertrophy, and agonists and growth factors are more involves in the pathological hypertrophies.
Hyperplasia: this is an increase in the number of cells. Physiological examples, which are usually hormone induced, include adrenal cortex due to stress, breast enlargement due to puberty and pregnancy, may coexist with hypertrophy such as the uterus in pregnancy. Pathological examples which can also be hormone induced, include the adrenal cortex due to a pituitary tumour secreting ACTH, enlargement of the prostate and endometrial hyperplasia, this could be considered as pre neoplastic is glands are atypical. There is also compensatory hyperplasia, for example in liver and kidney resection.
On the left, is a normal uterus and on the right is an enlarged uterus during pregnancy.
Epidermodysplasia verruciformis
This is an example of hyperplasia driven by a viral infection, in this case, HPV is the cause. There are obver 150 types of HPV, the most commonly known are 16 and 18 which cause cervical cancer, and 6 and 11 which cause warts. HPV 5 and 8 cause growths (seen in the photo below), others do also but these are the main contributors, the way they have affected
this man does suggest he was in an immunocompromised state. It is currently being researched how zinc and EVR1 and EVR2, genes which code for a zinc transport mechanism, affect HPV.
Metaplasia
This is a reversible change in which one cell type, epithelial or mesenchymal, is replaced by another cell type. This represents an adaptive substitution of cell types which are sensitive to stress with cells that are better to be able to withstand the different environment, which would cause stress or trauma on the original cell. This does not result from a change in the phenotype of a differentiated cell type, instead it is the result of a reprogramming of stem cells which are known to exist in normal tissues, or of undifferentiated mesenchymal cells present in connective tissue. In metaplastic change, these precursor cells differentiate along a new pathway. For example, the metaplastic transformation of oesophageal stratified squamous epithelium into mature columnar epithelium, which can be found in intestinal cells that cause acid reflux this is a pre neoplastic condition, this metaplastic change can develop into cancer if the stress/trauma to the cells is not removed. A risk factor towards this is smoking, although it is primarily caused by HPV, HPV itself can cause a variety of cancers, including cancer of the vulva, penile, oral, anal and skin cancer. HPV requires the presence of stratified squamous cells in order to grow.
Dysplasia
This is the term used in order to describe cells which have:
- An increased rate of cellular division often with abnormal mitotic figures
- Incomplete maturation
- Loss of normal cellular architecture
- Tend to have a high nuclear to cytoplasmic ratio
The presence and severity of dysplasia can be regarded as an indicator of the risk status of a precancerous lesion, and can be graded however this is very subjective, and so is basically the opinion of the the scientist observing it. Severe dysplasia indicates a very high risk of the subsequent development of cancer. Pre-neoplastic cells however can sometimes be reversible. Dysplasia is defined by 19 parameters.
Scrapes can be taken of the cells, however these are usually only on the surface of the epithelial layer. In cervical cancer, they can be categorised into cervical intraepithelial neoplasia 1, 2 and 3.
Histological changes in dysplasia diagnosis
- Loss of polarity of basal cells
- The presence of more than one layer having a basaloid appearance
- Drop-shaped rete-ridges (epidermal thickenings)
- Increased nuclear-cytoplasmic ratio
- Nuclear hyperchromatism
- Enlarged nucleoli
- Increased number of mitotic figures
- Mitotic figures that are abnormal in form
- The presence of mitotic figures in the superficial half of the epithelium
- Cellular and nuclear pleomorphism (variability in size and shape)
- Irregular epithelial stratification
- Loss of intercellular adherence
- Keratinisation of single cells or cellular groups in the prickle cellular layer
- Mild and severe dysplasia is easier diagnose than moderate
- If any of these changes become extreme, it can lead to cancer
- In cancer you get more than one nucleoli
There are 3 major problems attached to the importance of epithelial dysplasia in predicting malignancy development;
- There is a set of criteria for assessing epithelial dysplasia however the overall diagnosis is very subjective
- Not all lesions showing dysplasia will become malignant, some regress
- Carcinomas can develop from lesions in which epithelial dysplasia has not been diagnosed in previous biopsies.
In this case, there is a substantial requirement to improve the histological assessment of epithelial dysplasia, or, since it does not seem to be invariably associated with or even a necessity for malignant development, it may be necessary to develop other methods for predicting the malignant potential for premalignant lesions.
DNA ploidy analysis is a good prognostic marker, as it is objective, in comparison to histological grading, which is subjective.
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