Answers by yaoyufang

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									                        CANCER BIOLOGY MODULE
Answers to Private Study Questions
QUESTIONS:

Q1.   What are the clinical and histopathological features of cervical pre-
      malignancy and cervical cancer?

A1.   • Cervical pre-malignancy (cervical intraepithelial neoplasia, CIN) is generally detected
      by the cervical screening programme, when abnormal cytological changes are observed
      (e.g. increased nuclear/cytoplasmic ratio and mitotic figures). In the corresponding
      histopathological biopsy, these abnormal cellular changes can be found in the cervical
      epithelium. The extent of involvement of the epithelial layer gives the grading of the CIN
      lesion into high- or low-grade changes. In low grade CIN 1 lesions, the upper two-thirds
      of the epithelium shows good differentiation; in CIN 2/3 lesions, there is very little
      cellular differentiation throughout the epithelium.

      • CIN occurs in the transformation zone of the cervix. There are two types of cell found
      on the cervix, the flat squamous and the tall columnar types. Where these meet is the
      squamo-columnar junction (SCJ). The SCJ changes its position on the cervix under a
      number of hormonal influences. The area where cervical epithelial cells undergo the
      structural change between squamous and columnar shape (‘metaplasia’) is the
      transformation zone and it is where abnormal cellular changes (‘dysplasia’) leading to
      transformation occurs. Dysplastic changes can be observed using a special microscope
      (colposcope) and stains (acetic acid, producing ‘acetowhite’ areas).

      • The most common presenting features of cervical cancer are postcoital and
      intermenstrual bleeding. Pelvic and leg pain can occur during the later stages of the
      disease, due to tumour spread. Cervical tumour invasion frequently involves the bladder
      and rectum. Investigation is initially vaginal examination followed by a smear and punch
      biopsy. If the biopsy confirms cancer, staging involves rectal examination and
      cystoscopy. Magnetic resonance imaging gives the best estimate of tumour size but not of
      nodal spread which may only be found post-operatively at surgical dissection.

      • Most cervical carcinomas are squamous, only about 10% have a glandular structure
      (adenocarcinomas).


Q2.   What is the treatment for cervical cancer and cervical pre-malignancy?

A2.   • The primary treatment is different for cancerous and pre-malignant disease. Invasive
      cancer is treated by radical hysterectomy. In young patients, such treatment will try to


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      preserve the ovaries and vagina. If lymph node involvement or invasive spread is present,
      radiotherapy is the treatment given. Radiotherapy will also be given if lymph node
      involvement is found post-operatively. For low-grade CIN, a biopsy alone will be
      followed by an 80% regression of the lesion. Spontaneous remission of low-grade CIN is
      common. For high-grade CIN, which carries a high risk of progression to cervical cancer,
      treatment is local ablation via a loop excision or laser/diathermy of the transformation
      zone.

      • Radiotherapy treatment for cervical cancer can be both local (‘intracavitary’, where
      uterine cavity deposits and vaginal packs of radioactivity are applied) and more general
      (‘external beam’, where the tumour and lymph nodes are targeted at a distance from the
      radiation source). Either gamma rays (from radioactive isotopes, e.g. 137Cs, 60Co) or X-
      rays (produced by electrons in a linear accelerator) can be used to initiate tissue damage.


Q3.   What is the evidence that cervical cancer is caused by an infective
      agent? What are other potential causative factors of this disease?

A3.   • Earlier work noted that cervical cancer was extremely rare in women who had not had
      sexual intercourse (e.g. nuns). The incidence of cervical cancer is related to age at first
      intercourse and number of lifetime sexual partners of a woman. There are high rates
      among prostitutes.

      • The incidence is lower in women regularly using the diaphragm and/or barrier
      contraception in the partner. The larger the number of female partners a male has
      previously had, the increased the risk of cervical cancer in the current female partner. The
      risk is also increased for women whose partner has previously been married to a woman
      with cervical cancer. An association with other sexually transmitted diseases is also
      prominent. These factors point to a sexually transmitted infective agent and it is likely
      that this is human papillomavirus (HPV).

      • Other potential causative agents include smoking (although this may be related to
      sexual activity), diets low in certain constituents (β-carotene, vitamins C, E and folic
      acid) and other infective agents, e.g. herpes simplex virus type 2 (HSV-2). The incidence
      of cervical cancer is also influenced by immunosuppression (e.g. following renal
      transplantation), which potentiates both the frequency and severity of the disease, as well
      as infection with human imunodeficency virus (HIV).


Q4.   What is the structure of the human papillomavirus (HPV)? Which
      family members are associated with cervical cancer?




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A4.   • Papilloma and polyoma viruses are members of the Papovaviridae family. Though
      morphologically similar they are genetically different and each is now considered to be a
      distinct family. The human papillomavirus (HPV) is a small DNA virus. It contains
      circular, double-stranded DNA, about 8kb in length. The virus is un-enveloped and has
      icosahedral symmetry. As a result of these physical characteristics it is relatively
      thermostable and can remain infectious for long periods.

      • The genes of HPV code for early (E) proteins, involved in viral genome replication,
      and late (L) proteins, which form the structural components of the virus particle.

      • More than 70 different HPV types are now known. They are morphologically identical
      and, though there is antigen sharing between them, they are separated into different types
      by sequence difference. At least 40 of these infect the lower genital tract and often more
      than one type will be present at the same time, sometimes up to 4 types simultaneously.
      About 15 types have been associated with cervical carcinoma and are considered to
      comprise a group of ‘high risk’ viruses. In London, the most common types of this group
      are HPV 16 and HPV 18. Other HPVs are considered to be a ‘low risk’ group, associated
      with benign lower genital tract disease, e.g. genital warts (HPVs 6, 10, 11, 31, 35, 42,
      50). For the high risk HPVs, HPV 16 may be associated with cervical squamous cell
      carcinomas and HPV 18 with cervical adenocarcinomas. Case-control studies show very
      high relative risk of cervical cancer in association with HPV infection, although not all
      cases of cervical cancer are found to contain HPV.




Q5.   Which other viruses are associated with human cancers?

A5.   • Although viruses are associated with a number of animal tumours (relating mainly to
      retrovirus infection), there are only a few viral infections linked to human tumours. Such
      infections are summarised below.

Tumour virus                     Virus type          Genome size (kb) Human tumour
Human T-cell lymphotrophic       Retrovirus          9                Adult T-cell
virus (HTLV-1)                                                        leukaemia/lymphoma
Hepatitis B virus (HBV)         Hepadnavirus (small 3.2               Liver cancer
                                DNA virus)
Human papillomavirus (HPV)      Papillomavirus (small8                Cervical, anal,
                                DNA virus)                            laryngeal cancer
Epstein-Barr virus (EBV)        Herpesvirus (large DNA
                                                     ~200             Burkitt’s lymphoma,
                                virus)                                nasopharyngeal cancer
Kaposi’s sarcoma herpesvirus (KSHV)
                                Herpesvirus (large DNA
                                                     ~200             Kaposi’s sarcoma, primary
                                virus)                                effusion lymphoma




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      Hepatitis C, an RNA flavivirus, causes persistent hepatitis and may present with primary
      liver cell cancer. The mechanism of this is unknown.

      • There are 3 possible mechanisms by which human DNA viruses cause cancer:
      (i)     Chromosomal rearrangements, e.g. where the MYC oncogene is placed next to
              the immunoglobulin heavy chain gene, on a separate chromosome, leading to
              increased expression of this oncogene in B-lymphocytes; this has been found with
              Epstein-Barr virus infection.
      (ii)    Transactivation, where a viral protein may act to alter cell transcription
              (suggested for HBV- and HTLV-1-related tumours).
      (iii)   Cell cycle control, where viral proteins can interact with, and disable, cell cycle
              proteins (e.g. in HPV- and KSHV infections). Viral proteins that have this
              property have common amino acid sequence motifs that allow specific
              interactions with cell cycle proteins.


Q6.   By what mechanism can HPV infection lead to cervical cancer?

A6.   • There are common features and pathways to cell transformation for many of the DNA
      virus transforming infections. These are typified by KSHV and HPV in humans and by
      adenoviruses and polyoma viruses in animals. For a DNA virus to be able to transform
      cells the infection must not lead to cell death. Thus, viral replication is often halted before
      complete expression of the viral genome. Such infection is called ‘latent’ as opposed to
      ‘lytic’. Where an infection is linked to cell transformation, at least part of the viral
      genome will persist in the transformed cells. It can be shown that cell cycle control
      proteins, principally p53 and pRb, are bound up with, and inactivated by, viral proteins.
      It is known from studies of cancer trait families that genetic damage to these two control
      proteins will lead to a high familial incidence of cancer, thus inherited pRb loss of
      function leads to retinoblastoma in infants. Tumorigenesis by HPV, KSHV, adenoviruses
      and polyoma viruses is due to the binding of p53 and pRb by non-structural products of
      latent infection by these agents.

      • HPV infection is confined to epithelial cells. Complete viral replication and assembly
      occurs only in cells undergoing terminal differentiation. Viruses will reside in the
      germinal cells of the basal epithelium and replicate as multicopy episomal plasmids. In
      these basal cells the genome replicates once per cell cycle and this is faithfully
      partitioned into daughter cells, resulting in latent infection where only the early (E) non-
      structural proteins are expressed. In terminally differentiated squamous cells, no cellular
      DNA synthesis occurs, full viral particles, with capsid coats, are multiply produced and
      all viral genes (E and L) are transcribed; this is ‘productive infection’ which results in
      cell death (lysis). Viruses are then released and sexual transmission is possible between
      partners. At this point in the infection histological changes occur, including koilocytosis
      (irregular nuclei surrounded by a perinuclear ‘halo’).



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      • HPV infection in histological sections can be detected by a variety of molecular
      biological techniques, including immunohistochemistry, in situ hybridisation, Southern
      blotting, PCR and in situ-PCR. HPV similarly can be visualised in cervical smears. More
      commonly, HPV DNA can be detected by examining exfoliated cells taken during the
      smear test with molecular hybridisation assays (‘hybrid capture’).

      • The HPV genome contains early and late regions, depending upon the time course of
      transcription. The early E proteins are involved in viral DNA replication, the late L
      proteins are structural and form the capsid coat of the virus and are only transcribed in
      terminally differentiated cells. The segment of DNA between the end of late protein
      synthesis and early protein synthesis is the upstream regulatory region (URR), involved
      in the control of viral protein synthesis and replication. During long term and persistent
      infection the HPV genome may become integrated within a host cell. When this happens
      the integrity of the viral genome is disrupted and the circular HPV DNA is opened in the
      region encoding E1/E2 proteins. Since the E2 protein controls early gene transcription by
      binding to the URR, loss of E2 function leads to overexpression of the E6 and E7
      proteins. Often the early genes are the only viral sequences left in the HPV-induced
      cancer cells. Their presence is necessary for the maintenance of the transformed state of
      the cells.

      • The transforming ability of HPV 16 and 18 in cervical cancer samples is due to the
      strong binding of the E6 protein from these viruses to the cell cycle regulator p53,
      resulting in the rapid proteolysis and loss of function of this protein. In addition, the E7
      protein from these viruses strongly binds to pRb, the retinoblastoma protein, releasing the
      host cell factor E2F, which stimulates cellular growth.


Q7.   Why do you think the immune system cannot deal with the growth of
      cervical cancer cells?

A7.   • The immune system certainly has a role to play in the prevention of cervical cancer
      development, a virally-induced cancer. Patients on immunosuppressive therapy (e.g. for
      renal transplants) or those who are immunodeficient (primary immunodeficiency) have
      an increased risk of cervical cancer as well as benign disease (e.g. warts). Infected
      patients have CD4 T cell infiltrates in regressing warts.

      • In general, immune cells can recognise virus-specific antigens on tumour cells and
      these antigens are processed and presented in association with major histocompatibility
      complex (MHC-I) molecules, making them targets for cytotoxic T cells. However, only a
      small specific response will be generated as most of the ‘antigen’ on the tumour cells are
      ‘self’ (i.e. the immune system is tolerant to them).




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      • As with other tumours (e.g. breast, colon cancer), mechanisms of escape from immune
      surveillance occur in cervical cancer, including loss of MHC-I molecules, production of
      inhibitory molecules by the infected keratinocytes (including TGF and IL-10) and the
      fact that antibodies/cytotoxic cells have difficulty in accessing the surface of cervical
      tumour cells within a growing mass.



Q8.   How effective do you think the cervical screening programme is and
      how could it be improved?

A8.   • In the UK the cytology-based national screening programme for cervical cancer,
      together with the consequent treatment of pre-invasive lesions, has greatly reduced the
      mortality from this disease. Worldwide, cervical cancer incidence reflects the screening
      programmes, e.g. the incidence is high in South America (where there is no organised
      screening programme) compared to the U.K. Within Europe, cervical cancer incidence
      rates also reflect the use of the screening programme. For instance, cervical cancer rates
      have dropped in all Scandinavian countries (where cervical screening is now carried out)
      except Norway (where there is no such programme).

      • However, false negative results do occur and these are of particular concern as treating
      early versus late cervical cancer may significantly influence prognosis.

      • Despite the existence of the national screening programme, there were 3500 cases of
      invasive cancer of the cervix (with 1300 deaths) in England and Wales in 1995. Most of
      these were in women under 35 years of age.

      • The cervical cytological screening programme is based upon Papanicolou staining of
      cervical smears (‘Pap’ smear) to detect CIN. Screening every 3 years (for the age range
      20-60 years) is as effective as annual screening, as long as there is good patient
      compliance and good clinical follow-up. Improvements in the cervical cytology screening
      programme (to reduce the incidence of false negative smears) have included the use of
      brushes instead of wooden spatulas, optimisation of staining techniques, better training of
      technicians and re-screening of smears. Whilst these measures have improved the quality
      of the service there are further measures that can improve the reading of cervical smears.
      These include the development of liquid-based cytology preparations (here the cervical
      cells are sampled into a buffer instead of onto a slide) and automated screening of
      cervical smears. In the automated screening programme, 128 of the most abnormal cells
      are focused on using automated technology. This allows the screener to concentrate on
      these areas. Subjective judgements, however, are still required.

      • As infection with certain HPV sub-types (e.g. HPVs 16, 18, 33) are associated with the
      development of CIN and cervical cancer, women with ‘high risk’ HPV types have


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      relative risks of 40-180 for the development of high grade cervical disease. The inclusion
      of HPV DNA detection in the cervical screening programme may be useful. It will help
      by identifying women at risk with asymptomatic CIN1 or CIN2 infected with high risk
      virus, who are 'false negative' on cervical cytology. It will also help in the management of
      women with mildly abnormal smears by identifying those infected with high risk virus
      who should be followed up more closely. These benefits would enhance the national
      screening programme but would also have obvious financial implications.



Q9.   How could a knowledge of the viral causes of cervical cancer be used in
      the therapy and prevention of this disease?

A9.   • The crucial aspect in identifying an infective cause of a human cancer is that this then
      has the potential to allow control of the cancer within the population. If the infection can
      be controlled with, for example, a vaccine or with powerful anti-viral therapy the cause
      of cell transformation can be removed. Thus, in those countries where hepatitis B causes
      liver cancer implementation of national hepatitis B immunisation of newborn children
      has greatly reduced the incidence of liver cancer already. Understanding the
      epidemiology of the relevant virus infection can enable social and behavioural
      intervention to decrease the viral burden in the population.

      • Cervical screening programme modifications.
      Screening could potentially be made more effective by the introduction of tests to detect
      oncogenic HPV types. The presence of persistent oncogenic HPV types in repeat smears
      is indicates a need for further investigation and close follow-up.

      • Potential prophylactic and therapeutic vaccines for HPV infection.
      (i)     Animal studies have shown that immunisation with papillomavirus-specific
              proteins results in protection from subsequent infection and that some of these
              proteins may also be used therapeutically to induce regression of existing lesions.
              Thus, vaccines made from ground-up wart tissue have been used as both
              prophylactic and therapeutic measures against such papillomaviruses in animals.
      (ii)    The effector phase of the immune reaction against HPV antigens includes both
              antibody and cell-mediated immunity. In the antibody response there is a high
              titre of natural neutralising antibodies against the L1 and L2 proteins of HPV 16.
              Using recombinant DNA technology it has been possible to create virus like
              particles (VLPs) from the self-assembling capsid proteins (L1 and L2) of
              HPV. Such VLPs are potential prophylactic vaccine candidates and are currently
              undergoing clinical trials.
      (iii)   Therapeutic vaccines based on HPV E6 and/or E7 are also in early phase clinical
              trials.



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• Antiviral drugs such as interferons may also have a role in the therapy of this virus-
associated malignancy. These inhibit the growth of HPV-containing skin cells in culture;
this is via down-regulation of E6/E7 mRNA expression. As a result, interferons may be
useful in CIN 1 therapy. At present this remains a theoretical possibility rather than a
practical reality. Similarly, antiviral drugs such as the nucleoside analogues may inhibit
viral replication and could be applied topically.

• Measures to reduce transmission of the oncogenic virus include
(i)     Use of condoms to reduce sexual transmission of HPV
 (ii)   Reduction in the number of sexual partners to help minimise the risk of
        infection

Such measures may also reduce the risk of venereal and other diseases.




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