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Herpesviruses Slide Set

Nasopharynageal Carcinoma of Epstein-Barr Virus Infections

NASOPHARYNGEAL_CARCINOMA

NPC is a malignant tumour of the squamous epithelium of the nasopharynx. It is very prevalent in S. China, where it is the commonest tumour in men and the second commonest in women. The tumour is rare in most parts of the world, though pockets occur in N. and C. Africa, Malaysia, Alaska, and Iceland. The most undifferentiated form of the tumour is always associated with EBV whereas the rarer, more differentiated forms are not consistently so. The evidence that EBV is involved in the pathogenesis of NPC is as follows :-

1. Multiple copies of the EBV genome can be detected in the malignant cells of 100% of undifferentiated NPC. All the malignant cells express EBNA-1 and some have been reported to express LMP. Furthermore, infectious EBV particles can be recovered from NPC cell lines.

2. 100% of sera from undifferentiated NPC patients have high-titre antibodies to EB-viral antigens. As in BL, antibodies against VCA are at a 10 times geometric mean titre. IgG and IgA levels against EA (D) and VCA rise as the disease progress and may be used for screening and monitoring purposes. VCA and EA (D) IgA are also uniquely found in the saliva of NPC patients.
 

Cofactors - NPC is a genetically restricted tumour, being most common in the Southern Chinese, with intermediate frequency in some Negro and Mongoloid races and rare in Caucasians. Studies have shown that first-generation immigrants from S. China retain the high incidence of the disease, with the later generations showing a decline in incidence. This suggests that environmental as well as genetic factors are involved. NPC is especially associated with certain HLA haplotypes eg.HLA A2. More genetic linkage studies demonstrated the presence of NPC susceptibility genes near the MHC genes. Environmental factors are thought to play a role, in particularly the consumption of salted fish and foods containing nitrosamines.

Clinical Features - NPC occurs at a rate of 98 per 100,000 of the population of Southern China and is more common in men than women. The EBV associated undifferentiated type arises mainly in younger patients whereas the more differentiated types occur in older patients and constitute the bulk of the sporadic cases. The tumour most commonly arise in the posterior wall of the nasopharynx in the fossa of Rosenmuller, where it often remains silent and metastasizes to the local lymph nodes. The most common presentation of NPC is bilateral enlargement of the glands in the neck. The primary tumour may be very small and difficult to locate. Less frequently, the patient may present with the symptoms of invasion by the primary tumour eg. nasal obstruction, postnasal discharge, epistaxis, partial deafness and cranial nerve palsies. If untreated, the disease is rapidly fatal due to the development of laryngeal and pharyngeal obstruction.

Diagnosis - the diagnosis of NPC is usually made on histological examination of biopsy material. 3 types of NPC are recognized on histological appearance :-

1. a well differentiated SCC
2. a non-keratinizing carcinoma
3. an undifferentiated carcinoma

Serum antibodies to EBV antigens can be used to confirm the diagnosis and monitor the progress of the disease. Recent studies have demonstrated the value of testing for persistent high levels of serum IgA to VCA in screening for early lesions of the disease. It is hoped that early lesions can be diagnosed in this way and treated, and then monitored.

Treatment - NPC is difficult to treat surgically because of the early metastasis to regional lymph nodes. The tumour is resistant to chemotherapy and radiotherapy is the treatment of choice. However, because the tumour usually presents late, the prognosis is poor with a 5 year survival rate of 20%. It should in theory be possible to prevent the development of NPC with the use of an EBV vaccine at an early age.
 

EBV_Infection in the Immunocompromised

After primary infection, EBV maintains a steady low grade latent infection in the body. This latent infection is normally kept in check by the cellular defenses which, if impaired later in life, may lead to the reactivation of the virus to cause clinical disease. In a few cases, lymphoproliferative lesions and lymphoma may develop. These lesions tend to be extranodal and in unusual sites such as the GI tract or the CNS.

1. X-linked Lymphoproliferative Syndrome

Many families are known where the male members are unable to mount an effective cell-mediated immune response to EBV infection. The defective gene is located on the X chromosome. This condition accounts for half of the fatal cases of IM, the other half being sporadic with an equal age distribution. Clinically the affected male members are healthy until primary EBV infection occurs. The course of the disease can be either (i) fulminating and rapidly fatal, or (ii) progress on to a chronic phase which may culminate in a fatal B-cell lymphoproliferative disease, variously described as immunoblastic lymphoma, plasmacytoma and American Burkitt's lymphoma. These tumours often occur in the CNS or GI tract.

Many abnormal laboratory findings have been reported in X-LPS, including defects in NK cell activity and a defect in EBV specific T cell cytotoxicity. The pattern of antibodies to EBV antigens is also abnormal, with high titres of anti-VCA and EA antibodies and low EBNA antibodies. This pattern is identical to that seen in many immunosuppressed states and probably reflects increased viral replication leading to raised VCA and EA with decreased killing of infected cells resulting in low anti-EBNA titres. Female carriers show a milder derangement of antibody pattern but with normal EBV specific T-cell killing.

2. Transplant Recipients

Post Transplant Lymphoproliferative Disorder (PTLD) is thought to be a lymphoproliferation of EBV infected B-cells arising in the setting of over immunosuppression. The patients at risk are those who encounter EBV as a primary infection during the post-transplant course. The proliferation may be seen anywhere lymphoid tissue presides, although in lung transplant recipients, presentation in the allograft is relatively common. Histopathological manifestation appears as nodular sheets of atypical lymphoid cell which are not dissimilar to Non-Hodgkins lymphomas. Some cases are similar to lymphomatoid granulomatosis or T-cell rich B-cell lymphomas with a large subset of reactive T-cells. Reduction in immunosuppression often results in regression of PTLD.

It is well known that there is an increased incidence of malignancy following organ transplantation and maintenance immunosuppression. Malignant lymphoma accounts for 20% of such malignancies. Lymphomas have been reported to develop in 1 - 13% of patients following renal transplantation, 7.3% of cardiac transplants. Post transplant lymphomas have also been reported in bone marrow and heart/lung transplant recipients. The tumours tend to occur in the CNS, the GI tract, or in the transplanted organ. Most can be shown to carry the EBV genome and the cells express EBNA. High titres of antibodies to EA and VCA are present, which suggests increased virus replication. In around 50% of those developing EBV-associated lymphoma, there is evidence of recent primary infection.

It has been shown that the immunosuppressive therapy following transplantation reduces the CMI to EBV. Indeed, cessation of immunosuppressive had been accompanied by the regression of the lymphomas in some cases. Lymphomas in the immunocompromized are usually of the B cell type and classified as non-Hodgkin’s large cell lymphoma. They are usually extranodal, in the brain, gut and the transplanted organ and arise in 1-10% of transplant recipients. Around 80% of these tumours carry EBV genome and express EBNA. The large cell lymphomas express the same antigens as the lymphoblastoid cell lines, including the 2 viral oncogenes and cd 23. The majority of these tumours are monoclonal in nature.

AIDS

EBV-associated tumours occur frequently in patients with AIDS. Lymphomas are the second commonest malignancy in AIDS patients. However, not all lymphomas in AIDS patients are associated with EBV. The EBV associated tumours are (i) primary lymphoma of the brain and, (ii) Burkitt's lymphoma (50% of these tumours are similar to the African type and associated with EBV, whilst the other 50% are EBV genome negative). Recently, oral hairy leukoplakia has been described in HIV- seropositive individuals, forming multiple characteristic lesions on the lateral side of the tongue. DNA hybridization studies have shown active EBV replication in these lesions. In infants with AIDS, a lymphocytic infiltration of the lungs termed chronic interstitial pneumonitis in which the lymphocytes carry the EBV genome. However, it is thought that the syndrome is probably due to the direct action of HIV rather than EBV.
   

Laboratory_Diagnosis of EBV Infections

1. Infectious Mononucleosis - the classical finding in IM is the presence of atypical mononuclear cells in the blood. Lymphocytes would account for more than 50% of leucocytes present and of these, 20% are atypical. Atypical lymphocytes may also be seen in infections by CMV, hepatitis, influenza B, and rubella, but they are most prominent in IM. Abnormal LFTs are also present in the majority of patients. The diagnosis of IM may be suspected on clinical grounds and the findings of atypical lymphocytes, However, a firm diagnosis can only be made on serological testing. 2 types of serological tests are generally used for the diagnosis of IM.

The Heterophil antibody test is commonly called the Paul-Bunnel test, or the Monospot test (if done on slides). This test detects an antibody which causes agglutination of red blood cells from another species. False negative heterophil Ab test results occur and are more common in children under 14 years, especially under the age of 5 years. A possibility is that priming exposure to the unknown "heterophil antigen" has not taken place in very young children and thus no secondary response will arise on polyclonal stimulation. False positive heterophil Ab results are fewer in number than false negative results. Positive heterophil Ab results may last more than 6 months after the onset of IM and can occur in asymptomatic primary infection. This may be responsible for some of the "false" positive results.

IgM to VCA by indirect immunofluorescence is the best serological test available for the diagnosis of IM. However, this test is time consuming and results may vary between different laboratories. Also false positive results in the IgM may result from the cross linking between EBV specific IgG and anti-IgM conjugate by rheumatoid factor. If rheumatoid factor is present in the serum, it should be absorbed with staphylococcal protein A before testing. For these reasons, most laboratories rely mainly on the heterophil antibody test. A pre- illness specimen is rarely available to demonstrate a rise in IgG antibodies to VCA. High levels of VCA IgG are not diagnostic of acute infection. VCA IgM is generally used in the diagnosis of acute infection and IgG as an immune status screen.

Testing for EBNA antibodies may be of use in the window period. Anti-EBNA-1 antibodies do not usually arise until convalescence. Anti-EBNA-2 antibodies arise earlier in the illness and fall to low or undetectable levels during convalescence. The absence of EBNA- antibodies should not be regarded as diagnostic for IM as they are often undetectable in chronic IM, and conversely, they may be present soon after the onset of IM. Another possible confirmatory test is EBV-IgG avidity. The elution principle (avidity-index) is generally used for VCA-IgG.

2. Chronic IM - signs and symptoms of chronic IM range from fever, Pharyngitis, malaise, myalgia, and lymphadenopathy to potentially life-threatening problems such as anemia, thrombocytopenia, hupoglobulinaemia, and pneumonitis. Onset of chronic IM follows acute IM and may be due to impaired cell-mediated response to the virus. To meet the criteria for a diagnosis of chronic IM, three conditions need to be satisfied; (1) the symptoms should have persisted for at least 12 months (2) onset of persistent symptoms should have been preceded by a proven case of IM; (3) there should be evidence of active EBV infection . Highly elevated VCA and EA antibodies are often observed (>1024), EBNA-2 antibodies are frequently higher than those of EBNA-1 (the reverse of the situation is found in normal seropositive individuals), a positive heterophil antibody result may be seen, and more rarely, anti-VCA IgM may be detected. Similar antibody profiles have been observed in patients suffering from post-viral fatigue syndrome. It may be that some PVFS patients were, in fact suffering from chronic IM.

3. Burkitt’s Lymphoma - histology of biopsy specimens should reveal a poorly differentiated lymphocytic lymphoma. The tumour can be stained with antibodies to lambda light chains which should reveal a monoclonal tumour of B-cell origin. In over 90% of cases, the cells express IgM at the cell surface. The presence of EBV in tumour cells can be demonstrated by hybridization or the detection of EBNA-1. However, both these methods are technically demanding and therefore a diagnosis of BL is usually made on clinical and histological grounds. Children with BL have highly elevated titre of antibodies to EBV which may decrease following treatment and remission. Therefore, the determination of antibody levels may have a role in the monitoring of treatment. Although EBV serology might be of value in the early diagnosis of BL, such monitoring is not feasible on financial and practical terms.

4. Nasopharyngeal Carcinoma - the diagnosis of NPC is usually made on histological examination of biopsy material. However, the presence of EBV DNA and EBNA-1 can be readily demonstrated. Serum antibodies to EBV antigens can be used to confirm the diagnosis and monitor the progress of the disease, especially serum IgA to EA, VCA and ENA-1.. Recent studies have demonstrated the value of testing for persistent high levels of serum IgA to VCA in screening for early lesions of the disease and also for monitoring treatment.

5. X-linked Lymphoproliferative syndrome - the diagnosis of XLPS is much the same as for IM. In addition, EBNA-positive infiltrating lymphocytes can often be detected in post mortem or biopsy material. Serological studies of XLPS families commonly reveal a carrier state in healthy female relatives as evidenced by elevated EA and/or VCA titres. In such cases, genetic counseling may be given.

6. Post transplant lymphomproliferative disease - it is possible to detect EBV-DNA and EBNA in most of these lesions. The demonstration of EBNA positive cells is probably the most suitable method. Unlike BL and NPC, other latent antigens, ie. all the EBNAs and LMPA are expressed. The majority of EBV-associated post transplant lymphoproliferative lesions appear to occur following primary EBV infection. Serological diagnosis of primary infection is usually made retrospectively on sera taken for other purposes, as these patients rarely exhibit symptoms of IM. It may be possible to demonstrate a seroconversion. Patients who were seropositive prior to the transplant may have an antibody profile suggestive of reactivation.
   

VACCINE_DEVELOPMENT

A vaccine against EBV which prevents primary EBV infection should be able to control both BL and NPC. Such a vaccine must be given early in life. Such a vaccine would also be useful in seronegative organ transplant recipients and those developing severe IM, such as the male offspring of X-linked proliferative syndrome carriers. The antigen chosen for vaccine development is the MA antigen gp 340/220 as antibodies against this antigen are virus neutralizing. Inoculation of cotton top tamarins by purified gp 340/220 was able to protect the animals by subsequent virus challenge.
 

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Herpesviruses Slide Set