Ian Magrath, International Network for Cancer Treatment and Research
Published; Oct 2, 2012
Burkitt lymphoma (BL) occurs throughout the world and in all age-groups, but is the most common childhood malignancy (cancer) in countries close to the equator in Africa (and also in New Guinea), where it was discovered by an Irish surgeon, Denis Burkitt, in 1958. Here, it accounts for approximately half of all childhood cancers. Burkitt and his co-workers noted that the disease occurred particularly in warm wet regions, like other diseases known to be transmitted by mosquitoes (Yellow fever, for example). This suggested that insects, especially mosquitoes, may spread the disease. Children in equatorial Africa are invariably infected with at least two infectious agents, – Plasmodium falciparum, which causes malaria and is indeed spread by mosquitoes, and Epstein-Barr virus (EBV), which is spread by exposure to saliva from infected individuals, and is the cause of infectious mononucleosis. EBV is associated with a number of other illnesses, both malignant (e.g., nasopharyngeal cancer, Hodgkin disease) and non-malignant (e.g., a rare neurological disease resulting in paralysis called Guillane-Barré syndrome). Along with malaria, EBV it is believed to increase the likelihood that BL will develop. Infection with both probably occurs in the first year of life, particularly in rural regions. In fact, EBV infects essentially the entire population of the world, but the age at which infection is acquired, and its consequences, vary considerably in different world regions. In equatorial Africa, for example, one study showed that by the age of 3 years 100% of children have antibodies against EBV, indicating prior infection. EBV, however, is not known to produce any detectable illness at this age, although malaria, in contrast, is particularly deadly in infants and many die from the disease. Although infection with these two agents – a protozoan and a virus – is almost universal in equatorial Africa, the fact that only a small number of children develop BL indicates that the infections make it more likely, rather than a foregone conclusion, that BL will occur, which suggests that other factors, including chance, may also be relevant to the causation of BL. Other risk factors for BL, such as Arborvirus infections and a constituent of a plant that is widespread in Africa – Euphorbia tiruncalli – have been proposed on the basis of laboratory experiments, but they remain unproven. Even the mechanisms whereby malaria and EBV, respectively, could contribute to the development of BL have not been elucidated, although there are at least aspects of the biology of these microorganisms that enable scientists to suggest how they could increase the risk of developing BL. This is discussed in the following sections. In addition, whatever the factors are that contribute to the cause of BL, they seem quite potent insofar that the incidence of BL (the number of cases that occur each year in a given number of people, e.g., a million or 100,000) is perhaps 10-20 fold higher in equatorial Africa, compared, for example, to the incidence of BL in the USA or Europe. This is the reason that African BL is often referred to as endemic BL Denis Burkitt who worked in Mulago hospital in Kampala, Uganda, for many years after the second World War and where he recognized the clinical syndrome, that is, the pattern of involvement by BL of various parts of the body. Prior to his observations, published in 1958, African children with BL involving quite different parts of the body (see below) were generally thought to have different types of cancer. In equatorial Africa, the incidence of BL, is approximately 4 per 100,000 children less than 14 years old – similar to that of acute lymphoblastic lymphoma, which is the commonest childhood cancer in the USA or Europe. The incidence of BL in many countries outside Africa is unknown, but is likely to be somewhere between the relatively high incidence rates observed in equatorial Africa and the relatively low incidence rates found in the USA and Europe (1-3 per 1,000,000 children). BL is also predisposed to by immunodeficiency, whether acquired, as it may be in persons infected with the Human Immunodeficiency Virus, HIV or who have undergone organ transplantation, or inherited, as is the case in many diseases which impair immune reactivity in one way or another, such as the X-linked lymphoproliferative syndrome. Thus, the World Health Organization classification of hematopoietic malignancies refers to three different types of BL, endemic, immunodeficiency associated, and sporadic. The latter is the type that occurs in most of the world at lower incidence than BL in Africa, and without any known predisposing factors. The three types of BL have one thing in common, apart from their appearance, which is indistinguishable under the microscope. That is a molecular genetic abnormality known as a chromosomal translocation, in which two chromosomes in the cells destined to become BL cells exchange of parts of two (rarely, more) chromosomes. This is known as a chromosomal translocation, and in BL it most often involves one of the immunoglobulin genes (immunoglobulins are the proteins from which antibodies are made), which are situated on chromosomes 8, 2 and 22, and chromosome 8, which is the location of a very important gene known as MYC, which controls many vital molecular pathways in cells, including cell proliferation and the ability to survive in various environments cells encounter in the body. The translocation results in the inappropriate expression of the MYC gene, and this, along with other genetic changes which inhibit a very important cellular pathway (i.e., a series of molecular interactions within the cell) that causes it to destroy itself when it finds itself in places it should not be (a process known as programmed cell death, or apoptosis) is believed to be the cause of the BL cell’s abnormal behavior, i.e., giving it the ability to grow in places in the body that it shouldn’t. The need for a chromosomal translocation explains why malaria and EBV could not, by themselves, cause BL. They may increase the chances of a translocation developing (especially malaria), or be involved in the deactivation of the apoptotic pathways (especially EBV), but translocations are rare events, since they are highly dangerous to cells, and there are many checks and balances that exist in normal cells to prevent them from happening. Thus, BL, fortunately, is uncommon, even in Africa.
The meaning of “Aggressive” in the context of BL
BL is often described as “aggressive” because the tumor can increase in size very rapidly due to its potential doubling time of close to a 24 hours under optimal nutrient conditions (it is one of a handful of extremely rapidly progressive tumors). The doubling time varies according to the tissue that the tumor cells reside in as well being different in tumors in different individuals, so that some tumors are more aggressive than others. Occasional cases, e.g., of tonsillar BL, may remain apparently localized for long periods, although such cases are very rare indeed and one should always assume that a tumor diagnosed as BL is likely to have the ability to grow very rapidly. Because BL often infiltrates tissues (i.e., insinuates itself between cells) rather than growing as a lump, patients may sometimes conceal more tumor in their bodies than is immediately apparent, this can change rapidly, even in the course of only a few days. Thus, even when a patient appears to have only a small amount of tumor, it is safer to consider the detection of BL as signifying a potential medical emergency. It is important to rapidly establish a firm diagnosis, and to determine the extent of disease (which is relevant to the choice of treatment regimens) as quickly as possible, so that treatment can be initiated within a few days of reaching the hospital where he or she will be cared for. Since BL is a rare disease, most non-specialists see few, if any cases in a lifetime, and may not be aware of this. In situations where it is difficult to access medical assistance, such as in equatorial Africa, patients may die before they receive medical attention, or be moribund on arrival at a tertiary care facility.
Consequences of Rapid Cell Growth
The usually rapid growth rate of the tumor also means that new patients must be assessed as soon as possible for the presence or absence of emergent complications arising from the presence of tumor masses, such as breathing difficulties caused by compression of the trachea (wind pipe), intestinal obstruction, paralysis of the legs (paraplegia) and sometimes arms too (quadriplegia), or kidney failure, which can occur as a result of the very rapid cell division accompanied by a significant amount of cell death. This high cell turnover leads to high levels of uric acid in the blood in patients with extensive BL. Uric acid is not very soluble, and may precipitate in the kidneys causing physical blockage of the flow of urine which can rapidly give rises to dangerous increases in the blood level of potassium, which in turn can cause cardiac arrest and sudden death. Rapid infusion of fluid intravenously and administration of the enzyme rasburicase can reverse this dangerous situation, since rasburicase breaks down uric acid into the 5-10-fold more soluble substance allantoin, but if the level of potassium is high, other measures may need to be taken to reduce it as rapidly as possible while waiting for the urine flow to increase sufficiently to excrete the excess potassium. If rasburicase is not available (e.g., in Africa), patients with extensive BL and a raised serum level of uric acid need to be managed with allopurinol, a drug which prevents the formation of uric acid and is more readily available in lower income countries than rasburicase, and high infusion rates of intravenous fluids (good hydration is important in any event) to dissolve uric acid which may have already precipitated in the kidneys while slowing the ability of the body to produce more. If necessary, diuretics may be used to enhance the flow of urine, and to focus on maintaining (if possible) a high rate of urine flow during the first few days of treatment when the dissolution of the tumor due to treatment produces more uric acid (as well as phosphates, which can also cause blockage of the kidneys) so that the risk of kidney failure persists until a large portion of the tumor has been eliminated. Renal failure at the beginning of treatment, even if controlled, may necessitate modifications to chemotherapy, depending upon the treatment regimen being used, and every attempt to avoid it should be made, even if this means waiting an extra day or so before starting therapy to ensure that the uric acid level is close to normal and there is a high urine flow. Very often low doses of chemotherapy are given initially, to try to lessen the risk of massive tumor lysis, with high doses beginning a week later.
Cellular Origins of Burkitt Lymphoma
BL is one of perhaps 40 types of lymphoma (there is occasional disagreement with respect to what constitutes an individual pathological entity), i.e., it is a malignant tumor (cancer) of lymphoid cells, which comprise the most important element of the immune system. There are various types of lymphoid cell, the most numerous being the lymphocyte, present in all lymphoid tissue and comprising one of the types of “white cells” present in the peripheral blood. Some other cell types, such as macrophages, including dendritic reticular cells (see below), are also important elements of the immune system but are present in small numbers compared to lymphocytes and have something of an ancillary role, e.g., in storing antigen (a foreign substance, usually protein or carbohydrate, often derived from a microorganism) and presenting it to lymphocytes, or engulfing virus containing cells, or pieces of cells that have undergone apoptosis or lysis. Lymphomas arise as a consequence of genetic changes in their normal counterpart cells, or the cells which give rise to their normal counterpart cells. As described, the main genetic change in BL is the MYC-immunoglobulin translocation. Because there are many different types of lymphocyte, each with its more immature “parent cells” which continue to give rise to lymphocytes throughout life, and also many genetic changes that can give rise to malignant, or neoplastic disease (often simply called cancer, although technically, this is not the right word to use; neoplasm refers to a “new” growth, not normally present in the body), there are many different types of lymphoma. There is a considerable amount of information regarding both the nature of the genetic abnormalities present in BL cells and also the likely mechanisms that create them, in part based on mouse models and in part based on an interpretation of probable events based on the present understanding of how highly specific immune responses (adaptive immune responses) are generated. This topic will be addressed in more detail later. There is little or no knowledge, however, of what causes a tumor to arise in one individual versus another of similar age, lifestyle and environment, even in the immunodeficiency type of Burkitt lymphoma where the risk of developing the disease is generally higher than in the normal population. In high incidence areas it is inevitable that several cases may, from time to time, arise in close proximity to each other, both geographically and with respect to time, but with very rare exceptions where 2 or more cases occur in a family that is genetically predisposed, i.e., carries a mutation that makes it more likely that BL will develop in the offspring), there is no evidence that these are anything more than a chance, or random phenomenon. Many lymphoma types are more likely to occur with repeated exposure to environmental chemicals, such as agricultural pesticides, solvents and even hair dyes, but the majority of people who develop these diseases have had little or no exposure of this kind. Although, as mentioned in the first section, malaria appears to predispose to the development of BL in equatorial Africa, it is clearly not essential to the occurrence of BL, since this disease also occurs outside malarial endemic regions, although at a much lower frequency.
Distribution in the Body
The distribution of BL in the body is similar to that of certain lymphomas known to be caused by infectious agents (bacteria) – namely the benigh MALT lymphomas of the stomach which are associated with Helicobacter pylori (a type of bacterial infection), and similar lymphomas involving the tissues surrounding the eyes, which are associated with infection by another bacterium, Chlamydia psittaci. The involvement of the bowel and jaws (see below) could also be interpreted as being the sites of MALT (mucosal associated lymphoid tissue) which occurs in relationship to “mucous membrane” such as the lining of the bowel, and in various glands associated with the entire alimentary tract, from mouth to anus. This distribution pattern is more apparent in the equatorial African, i.e., the endemic form of the disease, in which the lachrymal glands, which make tears, and the salivary glands, as well as the thyroid and adrenals are not infrequently involved, although involvement of the jaw, nearly always in the developing molar teeth in young children, is the most frequent site of disease (occurring in 60% of patients). Essentially all African children aged 3 years have either jaw or orbital tumor, causing protrusion of the eyes. Orbital (the bony cave that protects the eye) involvement may well be one of the ways that BL enters the central nervous system (CNS), a complication which occurs in perhaps 15% of African patients at presentation, and more in patients with involvement of the orbits or maxilla (upper jaw). This is probably because it can invade the nerves that traverse the orbit, which it probably reaches via the maxilla . These “cranial nerves” are directly connected to the brain and are surrounded by a fluid (cerebrospinal fluid) filled sheath contiguous with the “sub-arachnoid” space – containing more of the cerebrospinal fluid – that surrounds the entire CNS, consisting of brain, spinal cord and cranial nerves. Reference to CNS involvement in BL usually means that there are tumor cells floating in the CSF, although rarely, solid masses of tumor may develop in the brain – usually only after prolonged involvement of the CNS and failure of therapy to eliminate it. The reason for the age association with jaw tumors in Africa children has never been elucidated, but most children have multiple tumors, often involving all four jaw quadrants, the tumor nearly always developing in and around the developing molar tooth buds and often invading the adjacent bone Throughout the world, the most common site of involvement is not the jaw, but the bowel, and up to 90% of patients with sporadic BL have disease involving the large or small bowel – even, occasionally, the appendix. In endemic tumors there is also a high degree of bowel involvement, but it is usually closer to 50%, albeit this could be an underestimate because of the lack of the expensive equipment required to identify disease not detectable by palpation of the abdomen. The areas of rather loosely aggregated lymphoid tissue (known as Peyer’s patches) that occurs at intervals along the inner bowel wall is often involved along with draining lymph nodes, although the latter may be enlarged, but may or may not contain tumor cells. The bone marrow (and sometimes involvement of peripheral blood) and liver are more often involved in sporadic tumors (i.e., outside Africa) than in endemic tumors, and jaw tumors are rare in sporadic tumors, even in young children. In fact, if they do occur, they usually cause relatively minor swelling of the gums and there seems to be no age association – even adults may have such tumors – although they are often associated with bone marrow and bony involvement. Lymph node involvement is somewhat variable, but certainly not as common as in most other lymphomas (in our recent series of 356 African patients, approximately 20% had peripheral lymph node involvement), which is not very different from the fraction of sporadic BLs with lymph node involvement. Involvement of the central nervous system (CNS) is relatively common, and extradural tumor (surrounding the cord) may result in paraplegia from either cord compression, which is potentially reversible if it has been present for only a short time (usually measured in days at the most) but usually permanent when infarction (death of spinal cord cells due to occlusion of their blood supply – the spinal arteries) has occurred. When CNS disease is present the cranial nerves, which govern movement and sensation in the head and neck region, are quite frequently involved, which can result in a variety of symptoms, including impairment of eye movements, paralysis of facial and neck musculature, occasionally affecting swallowing, and a loss of sensation in the facial region.
A Tumor of the Immune System – How Genetic Changes May Occur and Create a Lymphoma
BL is nearly always disseminated from the outset, i.e., spread to distant parts of the body, even if this is not obvious from the patient’s symptoms or examination by a doctor. Even special investigations involving X rays, or scans of various types may show no evidence of tumor other than that which led the patient to see the doctor in the first place. This is because the immune system – the system involved in protecting the individual against harmful effects of chemicals and microorganisms needs, itself, to be widely spread throughout the body in order to ensure that there are no gaps in the body’s defenses against harmful agents present in the environment. The immune system is made up of special cells that form highly structured cellular aggregates called lymph nodes, which are found in many parts of the body, especially…near points of entry for microorganisms or foreign substances. For example, lymph nodes are present in the neck and beneath the jaw. These often connect to the so-called Waldeyer’s ring, comprising the tonsils and the lymphoid tissue that encircles the throat – the point of entry for food and water which is frequently contaminated with viruses or other foreign materials that the immune system protects against. Although the stomach contains no lymphoid tissue, except in the presence of certain infections, there is a great deal of lymphoid tissue in the gastro-intestinal tract, including the Peyer’s” patches. The bowel lymphoid tissue is connected by fine lymph vessels to lymph nodes throughout the mesentery, the membrane by which the bowel is attached to the posterior abdominal wall. Lymph nodes are found in the axillae (arm pits) and inguinal regions (groins), since any microorganisms or other potentially toxic materials entering the body via a limb (e.g., as a consequence of infection occurring after an injury) would need to pass through these regions to reach the vital organs. Lymphoid tissue is also present in the form of lymph nodes at the so-called “hilar regions” of the lungs, where the trachea or windpipe divides into the two “main stem bronchi” that comprise the air passages of each lung. The hilar and other lymph nodes of the lung provide a barrier against airborne microorganisms or toxic substances. Finally, the blood is filtered by lymphoid tissue residing in the bone marrow and spleen and to a lesser extent, the liver. These locations are all sites where malignant tumors of lymphoid tissue can arise, although BL rarely involves the lungs, although it can cause quite large accumulations of fluid in the potential space between the lungs and the chest wall, known as the pleural space. Normally, the pleural membranes which cover the lungs and line the thoracic cavity provide small amounts of fluid to allow the lungs and chest wall to move freely over each other during normal breathing. Fluid can also accumulate in the pericardium, which surrounds the heart, or in the abdomen, from the lining which covers bowel and the abdominal wall – the peritoneum – which serves a similar purpose to the pleurae.
T and B cells
The immune system is divided into two major parts in which the lymphocytes have quite different functions. Bone marrow – derived, or B cells, make antibodies, and thymus derived, or T cells are more regulatory cells which circulate widely and interact with each other, with B cells and with other types of cell, e.g., macrophages. The latter are large cells with various functions ranging from engulfing microorganisms, dead cells, or part of cells, to processing proteins derived from microorganisms and “presenting” them to T or B cells, initiating the process of antigen recognition, whereby T or B cells and their progeny, are able to react specifically against the foreign material presented to them. Macrophages are able to produce a host of “cytokines” or chemical messengers that can recruit other cells to the site where the body has been invaded, and keep them there until the attempted invasion has been dealt with. Lymphoid cells ultimately arise from lymphoid stem cells. i.e., cells that have been derived from more immature and multipotential cells which reside primarily in the bone marrow, and which are capable of giving rise to lymphoid and myeloid stem cells (myeloid cells include the white cells of the bloodstream) as well as lymphoid stem cells. T cells subsequently migrate to the thymus, an organ in the upper part of the chest, where they undergo further differentiation into various lymphoid cell types which have specific functions, but in particular, regulate the immunological responses to foreign materials including microorganisms and may induce cells infected by microorganisms to undergo “programmed cell death” or apoptosis – i.e., to commit cellular suicide. T cells migrate throughout the tissues of the body, and provide a wide protective network. They are also present in all of the more formed structures (lymph nodes, spleen) where B cells are found, as well as in the bloodstream. The major function of B cells, in contrast, is the production of antibodies, which are immunoglobulin molecules able to bind to antigens. Antigens are usually part of a protein or carbohydrate derived from a microorganism or perhaps from the tissues of another animal or plant, which can be recognized by antibodies and determined to be foreign to the individual. When antibody binds to the microorganism it activates various processes that usually bring about the death of the microorganism, or sometimes simply “marks” the cell and assists T cells or macrophages to kill it through one means or another. The ability of antibodies to bind to antigens is initially very weak. Naïve B cells, i.e., lymphocytes that have not been previously exposed to antigen) express surface immunoglobulins that are able to react weakly to several antigens. They develop both a highly specific and strong ability to bind to antigen as a consequence of their passage through the germinal centers of lymphoid tissue. Antibody molecules are comprised of a variable region, which is the part responsible for antigen binding, and a constant region, which determines the more general characteristics (class) of the antibody – e.g., whether it will circulate in the bloodstream, or be secreted into saliva and various other body fluids, function primarily in the context of allergy, or be involved with various infections. Both the creation of antibodies with a high degree of specificity as well as the “switching” of the variable region to different constant regions, producing different classes of antibody with the same antigen specificity, occur in the germinal center. It is the activated naïve B cells themselves that create a germinal center in which they undergo several cycles of proliferation, during which the variable region (or most prominently, the “hypervariable” region) of the antibody molecule undergoes mutation, which results a modification of its ability to bind to the antigen. Cells making an antibody with a stronger affinity for the antigen are able to survive, but those with a weaker specificity cannot compete effectively for antigen, which is presented by macrophages residing in the germinal center, and are induced to undergo apoptosis. As they exit the germinal center, B cells with high affinity for antigen become either memory cells or plasma cells. Memory B cells are small resting lymphocytes which express their antibody at the cell surface, and survive for long periods of time, being reactivated either to a minor degree from time to time in order to retain the memory cell clone, or to a much greater extent if stimulated by the sudden presence of a large amount of the antigen their antibody is able to bind.
Burkitt Lymphoma – a B cell Neoplasm
Most lymphoid neoplasms arise from cells that are somewhere along the differentiation pathway from the stem cells of that particular tissue, to the most differentiated cell. Differentiation is the process whereby less mature cells give rise to more mature cells, e.g., lymphoid stem cells give rise to mature lymphocytes through multistep pathways that differ for T and B cells. The creation of an antibody molecule is both crucial to the adaptive immune response, and also a potential weakness in the B cell, since the DNA molecule must be broken to allow the various parts of the immunoglobulin molecule, which are physically separated on the chromosome, to join together to form an antibody. It is possible that some of the chromosome translocations (see below) present in BL occur as a “mistake” during this process. Both the induction of hypervariable region (i.e., regions within the variable region of antibody molecules which are particularly subject to mutation) is brought about by an enzyme called activation-induced cytidine deaminase. Artificial elevations of this enzyme in mice can bring about translocations involving immunoglobulin molecules and other genes. Thus, it appears very likely that the MYC-immunoglobulin translocations that are present in nearly all BL cells arise as a consequence of a “mistake” either during hypervariable region mutation or class switching. Such translocations may cause a change in behavior of the cell through the modification of the structure and function of genes adjacent to the translocation. Rarely, non-immunoglobulin genes are involved, and even more infrequently, there may be no translocation at all, in which case deregulation of the MYC gene appears to be accomplished by epigenetic phenomena (i.e., altered gene expression of the gene through modifications of its surrounding proteins, without any change in the DNA sequence). Although inappropriate MYC expression in normal cells causes apoptosis, if the pathways leading to apoptosis malfunction for any reason, the consequence is the creation of a BL cell. This is a crucial point, since it is possible that MYC-immunoglobulin translocations are arising in us quite frequently, as a result of the immunoglobulin recombination taking place in the germinal center but do not lead to malignant tumors since they switch on the pathways that lead to the death of the cell – a safety mechanism which has evolved to avoid the induction of a neoplasm. It seems highly likely that a failure of apoptosis permits the survival of cells which inappropriately express MYC. There are several possible mechanisms whereby this can occur. One involves a mutation in the MYC itself – such mutations are known to arise at the time of translocation. One such mutation is known to prevent the binding of a protein, known as BIM, to the MYC gene. BIM is necessary to initiate apoptosis when MYC is inappropriately expressed. Another possible mechanism is via Epstein Barr Virus genes – if they happen to be present in the cell.
Epstein – Barr virus and Burkitt Lymphoma
EBV was discovered in 1964 by Epstein, a pathologist interested in electron microscopy. He found evidence of the virus in cells derived from a BL that he received from Denis Burkitt, at that time, still in Uganda. Epstein was searching for a virus that might cause the tumor, since viruses were known to cause tumors in animals, and the distribution of BL in Africa was consistent with the possibility that it was caused by a virus vectored by a mosquito. At first, he was unable to find virus particles, but in one tumor, which took longer than expected to arrive from Africa, the cells had already disaggregated and were growing in the tissue culture fluid in the flask used to ship the tumor to Epstein. Epstein subjected the cells to electron microscopy and very quickly noted the virus particles present in a few of the cells. He recognized the virus as a type of herpes virus and suggested that it may be causally related to BL. Soon after, using cell lines sent to him by Epstein, Henle and his scientist wife, working in Philadelphia, developed a test for detecting antibodies against the new virus in the serum of various populations. He showed that most people in the USA are seropostive for the virus (their blood contains antibodies against it) but that children with BL in Africa have a much higher serum level of this antibody, which he called anti-virus capsid antigen (a capsid is a protein component of the virus itself). Henle developed more antibodies that were able to react with other viral proteins, e.g., those expressed when the cell is about to synthesize quite large quantities of virus (early antigens). He showed relationships between treatment outcome and the presence of anti-EA in the serum, including an increase in serum anti- EA levels immediately prior to relapse. Anti-EA antibodies were also shown to rise shortly after EBV infection, or immediately prior to the development of a tumor or recurrence. Subsequently, the virus life-cycle became quite well understood. EBV infects B cells, and travels with them through the germinal center in order to become a memory B cell, where it remains for the life of the infected individual, dividing infrequently, perhaps particularly in the nasopharynx from where the virus is released from lymphoid cells undergoing “lytic” infection (virus production) into the saliva. It is via saliva that EBV is usually spread from one person to another. In order to enter the bloodstream as memory B cells, virus-infected cells must cross the germinal center. Since virus could well infect cells that would not otherwise be activated, it needs to have mechanisms of preventing the cell from undergoing apoptosis during its passage through the germinal center – the fate of all cells unable to produce a high affinity antibody. A number of viral genes (known as latent genes, since they are expressed in that part of the virus cycle when virus particles are not produced), are known to be able to protect against apoptosis, and this too, therefore, may be a mechanism of preventing a germinal center cell which, by chance, develops a chromosomal translocation involving MYC, from undergoing apoptosis. Such an indestructible cell (at least within the context of the germinal center) is well on its way to becoming a BL cell. Interestingly, EBV is not present in all BL tumors, although around the world, the majority of tumors are EBV positive. In Africa almost 100% of tumors are positive, but in Europe and the USA, the figure is closer to 10%. It seems likely that malaria and EBV cooperate in causing BL. Malaria activates B cells- including those infected by virus, and causes them to increase dramatically in number – which is evident from the estimation of the number of circulating cells that contain EBV. This, increases the likelihood that a cell may both contain EBV and a MYC translocation. In HIV there is also an increase in circulating EBV DNA and virus infected cells, but interestingly, only about 30-40% of tumors are EBV+. The reason for this remains unknown..
Diagnosis – the Pathology of Burkitt Lymphoma
The usual method of diagnosing BL is to remove a piece of tumor, fix it in formalin (i.e., harden it), cut it into very thin sections that can be placed on glass slides, stain the cells with special dyes and look at them under the microscope at various degrees of magnification. When magnified markedly in this way, BL cells generally have a highly characteristic appearance (morphology), being round cells of intermediate size with a large central nucleus containing multiple nucleoli (2-5 areas where there is a lighter, rounded area within the nucleus). When stained by standard hematological dyes used to show up the morphological differences in various cell types, the cytoplasm is dark blue and contains many lipid vacuoles. Although, as cell populations go, BL cells are similar in size and shape, there are some tumors in which the cells are more variable than usual in that they contain a mixture of cell sizes, and may begin to resemble a related tumor called diffuse large B cell lymphoma (DLBCL). In some cases, it may be impossible to be certain, using standard microscopic and staining techniques, whether the tumor is a DLBCL or a BL and even highly experienced pathologists may disagree on the diagnosis. The pattern of protein expression by the cell (its immunophenotype), however, can be determined with the aid of monoclonal antibodies, which are carefully layered over the tissue on the slide, incubated then washed and looked at under the microscope. Because these proteins relate to the degree of differentiation of the cells, they are usually referred to as “cluster of differentiation” (CD) proteins. BL and DLBCL tend to have somewhat different patterns, but there is no absolute distinction between the two using immunohistochemistry alone. Both BL and DLBCL characteristically expresses B cell markers such as CD20 and CD79, as well as immunoglobulin – most often of the IgM class. BCL6 protein, which is involved in the creation of germinal centers is also expressed on both tumors, as is Ki67, a protein associated with rapid cell division. Ki-67 is expressed to a much higher level on BL cells than on DLBCL, – almost all cells express it, but some DLBCL express high levels of Ki67; sometimes more than 90%. BL nearly always expresses CD10, a molecule expressed on immature B cells (including B cell acute lymphoblastic leukemia cells) as well as the cells of the germinal follicle. CD10 is not characteristic of DLBCL, however, although some 10% or so of cases may express it. BCL2, a protein which protects against apoptosis is the converse, frequently expressed by DLBCL (and sometimes genetically rearranged at the BCL2 locus), and uncommonly by BL. Thus, a typical BL immunophenotype would be CD79a, CD20, CD10, BCL6 and Ki67, a typical DLBCL CD79a, CD20, and BCL6. Cells which have a more advanced degree of differentiation, and may resemble plasmablasts, tend not to express Ki67, but often express MUM-1 and CD138. For long, pathologists have recognized that some B cell lymphomas have a morphology that is not typical of either BL or DLBCL. These have been called variously, Burkitt-like lymphomas or atypical BL. The latter terminology is probably quite accurate because at the level of the gene expression pattern, these tumors are indistinguishable from BL. Thus, the morphology of BL is not well circumscribed. In fact some tumors which appear on morphology and immunohistochemistry to be typical DLBCL prove to have a gene expression pattern typical of BL (see below). Clearly, morphology, and immunophenotyping by immunohistochemistry, although the most widely used methods of diagnosis, are not 100% perfect. Fortunately, DLBCL particularly in young people (up to the age of about 30 years) is nearly always of the germinal center cell subtype of this kind of tumor, and treatment with standard BL therapy is known to be very effective. In fact in children and adolescents, this would be the treatment of choice for DLBCL. Thus, it is not critically important to distinguish between these two morphologies in young people. In older individuals, BL tends to be less common, and it not as certain that DLBCL in this age group responds well to “BL therapy” since such tumors are treated by medical oncologists rather than pediatric oncologists who generally use different treatment regimens. . In addition to study of the morphological and immunophenotypic characteristics of the cells, the chromosomal translocations present in the majority of BLs can be detected by a method known as fluorescent in situ hybridization. Briefly, the tumor cells are attached to glass slides and stained with probes consisting of DNA derived from a part or all of each of the genes involved in the translocations to which different colored fluorescent dyes have been attached (e.g., red for one gene and green for the other). Under appropriate conditions, the probes will bind (stick) to the genes present in the tumor cells. In normal cells, separated spots of each color can be seen, since the genes are on different chromosomes. In cells bearing a translocation the different colored spots are brought close to each other, or “fused” by the translocations. Two “fusions” can be seen – one for each of the chromosomes involved in the translocation, as well as separated spots of different colors where the probes have bound to the normal chromosomes (cells bear two copies of each chromosome, only one of which is involved in the translocation). BL cells may also be identified by an method known as molecular profiling, in which the pattern of expression of a large number of genes is examined. BL has been shown to have a distinct pattern, which differs markedly from the vast majority of cells diagnosed histologically as DLBCL. At present, this method is only carried out in research laboratories. The number of genes required to identify BL can, of course, be reduced to a minimum once the major differences between BL and related tumors such as DLBCL have been identified. Molecular or genomic profiling, has shown that the majority of morphologically atypical BLs and a small number of DLBCL have a molecular profile typical of BL. For now, the gene expression pattern is considered to be the final arbiter of the type of lymphoma. However, this method remains a research tool for the time being.
Chemotherapy is clearly the optimal treatment approach to BL, since purely local therapy (radiation or surgery), even if thought to be possible, which is not usually the case, very rarely leads to continuous complete remission in the absence of subsequent chemotherapy. This applies even to tumors the size of a sugar lump, which can sometimes be detected when they cause symptoms, e.g., tonsillar tumors, or small tumors in the small intestine, which may cause the bowel to invert, and unless spontaneous reversion occurs, result in swelling and intestinal obstruction. This condition, known as “intussusceptions” is extremely painful and nearly always prompts rapid surgical intervention. In this case, however, all apparent tumor is usually easily resected and such patients do extremely well even with only 2 or 3 cycles of unintensive chemotherapy. If, however, chemotherapy is not given, over 90% of such patients relapse and die. The treatment of Burkitt lymphoma in Africa was an early step in the development of modern treatment strategies. Burkitt himself, working with Burchenal from New York, and Clifford in Nairobi, showed that Burkitt lymphoma (almost all cases being children) responded to a number of anti-cancer drugs. Among them, cyclophosphamide, vincristine and methotrexate were shown to be particularly efficient. Over a period of several years it was observed that patients sometimes responded to different drugs if the drug given first proved to be ineffective or response was very short lived. In Uganda, this led to using drugs in previously defined sequences, sometimes with two drugs at a time, e.g., the regimens known as BIKE, consisting of cyclophosphamide followed by a combination of methotrexate and vincristine, or TRIKE, consisting of three cycles of treatment – cyclophosphamide followed by methotrexate and vincristine followed by cytarabine. Eventually, three drugs, cyclophosphamide, vincristine and methotrexate (COM) were compared to cyclophosphamide alone. Because patients were known to have a high likelihood of relapse in the CNS, the value of craniospinal irradiation to prevent CNS relapse was explored. It proved to be entirely ineffective. In Ghana another combination chemotherapy regimen was used. This included cyclophosphamide, vincristine and cytarabine (CVA) along with direct injection of methotrexate into the spinal fluid (intrathecal therapy, IT) in an attempt to prevent CNS relapse. The results with CVA were also compared with those achieved with cyclophosphamide alone given in an earlier time period and shown to be superior. Although these early studies were often conducted with rather few patients, there appeared to be survival advantages to the drug combinations in terms of the survival rate, although the CVA regimen was associated with considerably more toxicity, especially in the first cycle of therapy. These African contributions, extending over somewhat more than a decade, formed the backbone of modern therapeutic approaches the world over. They demonstrated the activity of a number of drugs, indicated the importance of CNS treatment (nearly always separate injections of cytosinie arabinoside and methotrexate into the spinal fluid) to prevent or treat CNS disease, and showed the value of combinations of drugs rather than single agents. In the USA and Europe, slight modifications of the regimens developed in Africa were used at the National Cancer Institute for both children and adults, but patients elsewhere were treated with regimens similar to those used either for other lymphomas, in the case of adults, or for patients with acute lymphoblastic leukemia in the case of children – at that time, the difference between leukemia and lymphoma was unclear, and lymphomas were considered to be identical to leukemias except that the bone marrow was not involved. As it happens, this is very close to the truth, although the ability to distinguish between different subtypes of lymphoma was poor, since it depended purely upon morphological comparisons. Eventually, a study was performed in children in which a simple approach derived from BL treatment in Africa was directly compared to therapy used for acute lymphoblastic leukemia in the USA, by assigning patients with advanced disease at random to either COMP (cyclophosphide, vincristine, prednisone and methotrexate) or to LSA2L2, a regimen modified from a treatment protocol used for patients with acute lymphoblastic leukemia (both regimens included intrathecal therapy). Patients with Burkitt lymphoma had a better outcome when treated with COMP than with LSA2L2. . These early studies in children were associated with long term survival results in the range of 50% – not greatly different from the results achieved in Africa. German, French and US investigators therefore studied the efficacy of much more intensive regimens. Adriamycin was added to the basic COMP regimen, and the dose of methotrexate elevated without a major benefit being observed, while other drugs, including ifosfamide and mesna (the latter to prevent hematuria, a common side effect of ifosfamide), cytarabine and etoposide were tested firstly in patients who had relapsed, and when shown to be active, brought into front line therapy. These more complex, and more intensive combinations give excellent results – approximately 80-90% long term survival, although the outcome in patients with CNS disease was somewhat worse. However, toxicity overall – both early and late (effects on fertility, the heart and second malignancies) was increased such that in recent years the major goal of clinical research on treatment has been to reduce the intensity of combination regimens while retaining their high degree of efficacy. The role of adriamycin has never been proven; it was added largely because of its value in adults with lymphoma, but this drug can cause both early and late cardiac toxicity, the latter even when a relatively low total dose of drug is given. Thus, its value is being tested as part of the continuing “fine tuning” of therapy. Several sequentially performed studies indicated that the intensive LMB regimens used in France for children with BL could be shortened and simplified and current regimens for children in France, the UK and the USA are based on a modified LMB regimen which is much shorter than the original regimen, but equally, or more efficacious. In adults, regimens based on successful regimens designed for children were eventually introduced with clearly better results. The regimen known as CODOX-M/IVAC, a descendent of the regimens used in Africa, with the addition of the drugs shown to be active in patients with recurrent disease was used in both children and adults in a small group of institutions led by investigators at the National Cancer Institute, and continues to be used, sometimes in modified form, for adults. The original regimen was associated with severe neuropathy in some patients treated with colony stimulating factors to reduce the duration of neutropenia. This side effect can be easily prevented without loss of efficacy by lowering the dose of vincristine. The possible role of a biological agent active in many B cell lymphomas, rituximab, is also under study. Results in adults suggest that it improves the survival of high risk patients, including those with HIV associated BL. Rituximab does, however, cause an increased rate of infection and it will be important to determine whether the doses of other agents can be reduced without loss of efficacy when rituximab is included in the regimen. It may prove to be a valuable addition to patients at highest risk for recurrence, namely patients with CNS disease, but at present it is still too expensive to be used routinely in the low and middle income countries. Because of the success of modern, intensive drug combinations, it has become more and more difficult to treat patients who relapse. Very high dose therapy followed by stem cell rescue (to replenish the stem cells destroyed by very intensive conditioning regimens in order to permit the patient’s blood cell counts to recover) have been occasionally successful, although there is a limited amount of information because of the relative rarity of recurrent disease. There is hope, but limited information, that when allogeneic stem cells (i.e., derived from another individual) are used, lymphoid cells derived from the stem cells may react against and destroy the remaining tumor cells. However, because of the fact that there are relatively few patients who relapse, and there is a great deal of variability in approaches to treatment of relapse, the value of very high dose therapy with stem cell rescue remains uncertain, although the lack of clear alternatives leads many oncologists to try this approach in patients with recurrent disease who still show significant response to salvage therapy (in those who show no response to treatment after relapse, or progress, there is essentially no chance of a good outcome). In addition, the definition of relapse has become an important question, since large tumor quite frequently leave scar tissue or dead tissue behind them. These residual masses may have the appearance of tumor on, for example, computerized axial scans, or PET (positron emission tomography) scans. Such scans are non-specific, however, and PET scans may be falsely positive when inflammatory cells are present in the residual mass. This may account for at least some of the apparent successes with intensive therapy and stem cell infusion – in fact, there was no residual disease. In equatorial Africa, and other countries in low and middle income groups, intensive therapy is rarely affordable or feasible, since limited resources lead to sub-optimal supportive care. In equatorial Africa, therefore, cyclophosphamide, vincristine and methotrexate, with IT therapy is widely used, although other regimens using higher intensity cyclophosphamide has also been explored. With such regimens survival rates are generally between 50 and 60% after several years, although this result may be improved upon if patients could be identified and treatment initiated at an earlier time in the course of the disease; at present the cost of transportation to an often distant tertiary care center and delays in making the diagnosis often result in the patient’s having very extensive disease at the time of diagnosis. In all, Burkitt lymphoma has led to many new ideas both with respect to how the molecular changes (in lymphomas, at least), arise, how environmental agents, such as malaria and Epstein Barr virus may contribute to pathogenesis, and how resistance to chemotherapy can be overcome by the use of multiple agents. It is likely that continued attempts to improve our understanding of BL will eventually lead to much simpler approaches to treatment, using molecular “targets” present in the tumor cells such that therapy is much more specific for the tumor cells than is the case when conventional cytotoxic drugs are used. Novel treatment approaches for EBV+ BL have also been explored in the laboratory. Given the excellent results currently being obtained, at least in the technologically advanced countries, the major value of such approaches would be reduced toxicity and the length of time likely to be needed to define and explore the value of targeted therapy makes it unlikely that such novel approaches will be introduced in the near future, although there is good reason to believe that improved survival will be achieved in the near future via the “fine-tuning” approaches discussed above. It is sad that most patients with BL die, many without any therapy, or after partial therapy; since Africa has contributed so much to the understanding and treatment of this disease, it would seem appropriate to focus more attention on the plight of the poor on this continent.