IMMUNODIAGNOSIS AND IMMUNOTHERAPY OF INFECTIONS (AMB 710)

BY CHUKWUBIKE, CHINEDU MB

REG NO: 2010487008P

DEPARTMENT OF APPLIED MICROBIOLOGY AND BREWING

FACULITY OF BIO-SCIENCES NNAMDI AZIKWE UNIVERSITY, AWKA

LECTURER: PROF. CHRIS ANYAMENE

PRINCIPLES OF IMMUNDIAGNOSIS AND IMMUNOTHERAPY

1

Immunological tests have the dual capacity to be used for diagnosis of recent infection or determining a patients immunity states. Both structural and non-structural components of bacteria, virus, fungi and other microorganisms usually induce antibodies during infection. Over the years many traditional immunological methods available and are: Agglutination Agglutination is the visible aggregation of particles caused by combination with specific antibody. Antibodies that produce such reactions are often called agglutinins. Because this reaction takes place on the surface of the particle, antigen must be exposed and able to bind with antibody. Agglutination is actually a two-step process, involving sensitization or initial binding followed by lattice formation, or formation of large aggregates. Types of particles participating in such reactions include erythrocytes, bacterial cells, and inert carriers such as latex particles. Each particle must have multiple antigenic or determinant sites, which are Cross-linked to sites on other particles through the formation of antibody bridges. Agglutination reactions can be classified into several distinct categories: direct, passive, reverse passive, agglutination inhibition, and co-agglutination. Principles of each of these types of reactions are discussed, including their current use in todays clinical laboratory STEPS IN AGGLUTINATION Sensitization Agglutination is a two-step process that results in the formation of a stable lattice network. The first reaction involves antigenantibody combination through single antigenic determinants on the particle surface and is often called the sensitization step. This initial reaction follows the law of mass action and is rapid and reversible. The second step is the formation of cross-links that form the visible aggregates. This represents the stabilization of antigenantibody complexes with the binding together of multiple antigenic determinants. Each stage of the process is affected by different factors, and it is important to understand these in order to manipulate and enhance end points for such reactions. Sensitization is affected by the nature of the antibody molecules themselves. The affinity and avidity of an individual antibody determine how much antibody remains attached. The class of immunoglobulin is also important; IgM with a potential valence of 10 is over 700 times more efficient in agglutination than is IgG with a valence of 2. The nature of the antigen-bearing surface is also a key factor in
2

the initial sensitization process. If epitopes are sparse or if they are obscured by other surface molecules, they are less likely to interact with antibody. TYPES OF AGGLUTINATION REACTION Direct Agglutination Direct agglutination occurs when antigens are found naturally on a particle. One of the best examples of direct agglutination testing involves known bacterial antigens used to test for the presence of unknown antibodies in the patient. Typically, patient serum is diluted into a series of tubes or wells on a slide and reacted with bacterial antigens specific for the suspected disease. Detection of antibodies is primarily used in diagnosis of diseases for which the bacterial agents are extremely difficult to cultivate. One such example is the Widal test, a rapid screening test to help determine the possibility of typhoid fever. The antigens used in this procedure include Salmonella O (somatic) and H (flagellar) antigens. A significant finding is a fourfold increase in antibody titer over time when paired dilutions of serum samples are tested with any of these antigens. While more specific tests are now available, this test is still considered useful in diagnosing typhoid fever in developing countries, and it remains in use in many areas throughout the world.

Passive Agglutination Passive, or indirect, agglutination employs particles that are coated with antigens not normally found on their surfaces. A variety of particles, including erythrocytes, latex, gelatin, and silicates, are used for this purpose. The use of synthetic beads or particles provides the advantage of consistency, uniformity, and stability. Reactions are easy to read visually and give quick results. Particle sizes vary from 7 m for red blood cells down to 0.8 m or less for fine latex particles. Many antigens, especially polysaccharides, adsorb to red blood cells spontaneously, so they are relatively easy to manipulate. Problems encountered with the use of erythrocytes as carrier particles include the possibility of cross reactivity, especially with heterophile antibody if the cells used are nonhuman. Passive agglutination tests have been used to detect rheumatoid factor; antinuclear antibody occurring in the disease lupus erythematosus; antibodies to group A streptococcus antigens; antibodies to Trichinella spiralis; antibodies to Treponema pallidum; and antibodies to viruses such as cytomegalovirus, rubella, varicellazoster, and HIV-1/ HIV-2.
3

Reverse Passive Agglutination In reverse passive agglutination, antibody rather than antigen is attached to a carrier particle. The antibody must still be reactive and is joined in such a manner that the active sites are facing outward. Adsorption may be spontaneous, or it may require some of the same manipulation as is used for antigen attachment. This type of testing is often used to detect microbial antigens. Shows the differences between passive and reverse passive agglutination. Numerous kits are available today for the rapid identification of antigens from such infectious agents as group B streptococcus, Staphylococcus aureus, Neisseria meningitides, streptococcal groups A and B, Haemophilus influenzae, rotavirus, Cryptococcus neoformans, Vibrio cholera 01, and Leptospira. Rapid agglutination tests have found the widest application in detecting soluble antigens in urine, spinal fluid, and serum. The principle is the same for all these tests: Latex particles coated with antibody are reacted with a patient sample containing the suspected antigen. In some cases, an extraction step is necessary to isolate antigen before the reagent latex particles are added. Organisms can be identified in a few minutes with fairly high sensitivity and specificity, although this varies for different organisms. For example, the sensitivity of latex agglutination kits for the detection of cryptococcal antigen has been reported to be as high as 99 percent, while the specificity of testing for Candida albicans is much lower Agglutination Inhibition Agglutination inhibition reactions are based on competition between particulate and soluble antigens for limited antibody-combining sites, and a lack of agglutination is an indicator of a positive reaction. Typically, this type of reaction involves haptens that are complexed to proteins; the haptenprotein conjugate is then attached to a carrier particle. The patient sample is first reacted with a limited amount of reagent antibody that is specific for the hapten being tested. Indicator particles that contain the same hapten one wishes to measure in the patient are then added. If the patient sample has no free hapten, the reagent antibody is able to combine with the carrier particles and produce a visible agglutination. In this case, however, agglutination is a negative reaction, indicating that the patient did not have sufficient hapten to inhibit the secondary reaction. Either antigen or antibody can be attached to the particles. The sensitivity of the reaction is governed by the avidity of the antibody itself. It can be a highly
4

sensitive assay capable of detecting small quantities of antigen. Detection of illicit drugs such as cocai ne or heroin. To perform a hemagglutination inhibition test, patient serum is first incubated with a viral preparation. Then red blood cells that the virus is known to agglutinate are added to the mixture. If antibody is present, this will combine with viral particles and prevent agglutination, so a lack of or reduction in agglutination indicates presence of patient antibody. Controls are necessary, because there may be a factor in the serum that causes agglutination, or the virus may have lost its ability to agglutinate. Coagglutination Coagglutination is the name given to systems using bacteria as the inert particles to which antibody is attached. Staphylococcus aureus is most frequently used, because it has a protein on its outer surface, called protein A, which naturally adsorbs the fragment crystallizable (FC) portion of antibody molecules. The active sites face outward and are capable of reacting with specific antigen .These particles exhibit greater stability than latex particles and are more refractory to changes in ionic strength. However, because bacteria are not coloured, reactions are often difficult to read. Such testing is highly specific, but it may not be as sensitive for detecting small quantities of antigen, as is latex agglutination. Coagglutination reagents have been used in identification of streptococci, Neisseria meningitidis, Neisseria gonorrhoeae, Vibrio cholera 0139, and Haemophilus influenzae.

RADIOIMMUNOASSAY The first type of immunoassay developed was radioimmunoassay (RIA), pioneered by Yalow and Berson in the late 1950s. It was used to determine the level of insulinanti-insulin complexes in diabetic patients. A radioactive substance is used as a label. Radioactive elements have nuclei that decay spontaneously, emitting matter and energy. Several radioactive labels, including 131I; 125I; and
5

tritiated hydrogen, or 3H, have been used, but

125

I is the most popular. It has a half-life of 60

days, and because it has a higher counting rate than that of 3H, the total counting time is less. It is easily incorporated into protein molecules, and it emits gamma radiation, which is detected by a gamma counter. Very low quantities of radioactivity can be easily measured. RIA was originally based on the principle of competitive binding. Thus, the analyte being detected competes with a radiolabeled analyte for a limited number of binding sites on a high-affinity antibody. The concentration of the radioactive analyte is in excess, so all binding sites on antibody will be occupied. If patient antigen is present, some of the binding sites will be filled with unlabeled analyte, thus decreasing the amount of bound radioactive label. When bound and free radiolabeled antigens are separated and a washing step has occurred, the amount of label in the bound phase is indirectly proportional to the amount of patient antigen present. . Advantages and Disadvantages of Radioimmunoassay Examples of substances that are measured by RIA include thyroid-stimulating hormone and total serum IgE. RIA is an extremely sensitive and precise technique for determining trace amounts of analytes that are small in size. However, chief among the disadvantages of all RIA techniques is the health hazard involved in working with radioactive substances. Laboratories have found it more and more difficult and expensive to maintain a license and to comply with federal regulations. In addition, disposal problems, short shelf life, and the need for expensive equipment has caused laboratorians to explore other techniques for identifying analytes in low concentration. Enzyme immunoassays have largely replaced RIA because of their comparable sensitivity and the availability of automated instrumentation that allows for processing of a large number of samples in less time

Enzyme Linked Immuno Sorbant Assay (ELISA) Enzyme Linked Immuno Sorbant Assay (ELISA) also called EIA (Enzyme Immunosorbant Assay) is a very sensitive and safe technique used in the detection of antigens and antibodies was introduced by Engvall and Perlmann. The sensitivity of this technique is comparable with that of Radio immunoassay with an added advantage of safer use of non radioisotopic reagents and longer shelf life of the same.
6

It also eliminates the requirement of sophisticated isotope counters. ELISA can be done in smaller laboratories and is adaptable to field conditions as well. The application of ELISA has been well reviewed. ELISA plays an important role in the laboratory and in the field. The various components involved in ELISA are a solid phase to which specific antigen/antibody is coated, an antigen or antibody enzyme conjugate as probe as the case may be and enzyme substrate. Solid supports used are polystyrene or PVC microtitre plates, tubes or beads. Nitro cellulose paper or Cellulose Acetate Membrane are also used as solid phases. Enzymes used for conjugation to anti immunoglobulins include peroxidase, b-galactosidase, alkaline phosphatase, penicillinase, urease, glucose oxidase. No enzyme fulfills all the criteria for an ideal label in EIA. The influence of the solid phase on the enzyme should be minimal. Conjugation to anti-immunoglobulin should be easy and conjugates should be active and stable. These are the reasons for frequent selection of peroxidase in commercial diagnostics. In our laboratory we have been successful in using penicillinase as marker in EIA for tropical diseases. Penicillinase has also been successfully used as an enzyme marker in hormone assays. Penicillinase (blactamase, EC 3.5.2.6) has high turnover number of 1, 60,000 and has been found to be more sensitive than peroxidase, alkaline phosphatase or b- galactosidase. The substrate (Penicillin V) used is not carcinogenic and other advantages of using penicillinase are that the enzyme is quite stable and is not present in biological fluids under normal conditions. Penicillinase activity can be reliably estimated iodometrically. The assay involves decolourisation of blue coloured starch-iodinepenicillin substrate. The result is assessed visually by naked eye. The reaction may be stated as follows. Indirect ELISA Indirect ELISA is useful for the detection of antibody using specific antigen say filarial antigen. In this assay, the PVC plate was coated with antigen and the test sample (serum/blood, hydrocele fluid, urine, etc.) was added to the plate. Any antibody specific for the antigen will bind to the available sites. The bound antibody was detected by incubation with an enzyme labelled specific anti-immunoglobulin followed by the enzyme substrate.
7

Indirect ELISA using W bancrofti mf ES antigen, incubated with filarial sera samples followed by anti-human IgG-penicillinase conjugate and starchiodine penicillin substrate was found to be highly sensitive in detection of filarial infection. As little as 0.35 ng antigen protein per well was found to be sufficient in detecting filarial antibody compared to 0.1-1.5 g of soluble mf antigen protein per well used earlier. Direct ELISA Direct ELISA was used in the laboratory for detection of antigen as in filarial immunecomplexes. Optimal dilution of circulating immunecomplexes (10 g/ ml) was coated on to the plate. After washing, filarial serum immunoglobulin Gpenicillinase (FSIgG-penicillinase) conjugate was added and then assayed. Analysis of immune complexes showed the presence of filarial antigen in 30 out of 33 clinical filarial, 2 out of 15 endemic normal and none of the non endemic normal sera.

Competitive ELISA Competitive ELISA is useful for identification and quantitation of either antigen or antibody. In antigen determination by this method, antigen present in the sample competes for sites on the antibody with labelled antigen added to the medium. The colour change will be inversely proportional to the amount of antigen in the sample. Competition principle can be exploited in number of ways. To identify the antigen involved in filarial immunecomplexes, circulating immunecomplex (CICs) showing filarial antigen in direct ELISA was
8

coated on to the plates. Then 50 l of culture fluid containing W. bancrofti mf ES antigen (7ng/ml) was added together with 50 l of FSIgG penicillinase conjugate. After incubation and washing, the enzyme activity was assayed. In this assay system, freely added W. bancrofti mf ES Ag competes with the immune-complexed antigen bound to the plate, for binding sites on FSIgG conjugated with penicillinase. Presence of W. bancrofti mf ES antigen in CICs was confirmed by the persistence of blue colour

Sandwich ELISA Double antibody sandwich ELISA is useful for detection of antigen. In the assay system the antigen to be detected is sandwiched between two similar or different antibodies of which one is labelled with an enzyme. IgG fraction of human filarial serum immunoglobulin (FSIgG) has been successfully used for detection of circulating filarial antigen by sandwich ELISA. When analysed using FSIgG sandwich ELISA, 27 out of 33 microfilaraemia, 19 out of 30 clinical filarial and none of the 20 non-endemic normal sera showed the presence of filarial antigen. Detection of filarial antigen using FSIgG in sandwich ELISA showed an apparent positive correlation between microfilarial density and antigen titre. When Wb E34 monoclonal antibody was used along with FSIgG in double antibody sandwich ELISA 68% of microfilaraemic sera showed the presence of filarial antigen. Detection of filarial antigen in urine and hydrocele fluid samples by sandwich ELISA using FSIgG was also reported. Stick sandwich ELISA was also developed for detection of circulating free antigen and CIC-antigen in tuberculosis sera Inhibition ELISA
9

Inhibition ELISA works similar to competitive ELISA but in this system the two antigens (antigen in test sample and enzyme labeled antigen) are added one after another. This is useful especially when test serum contains both antigen and antibody of interest in the immune reaction. Inhibition ELISA was also useful in determining the identity of specific antigen or antibody

Anti C3 ELISA: Anti C3 ELISA is useful to detect disease specific circulating immunecomplexes or specific antigen present in circulating immune-complexes. Test sera are incubated in micro titre plate wells prior sensitized with rabbit/goat anti human C3 antibody. The filarial antigen in bound immunecomplexes is detected by further incubating the wells with enzyme labeled specific antibody. In our laboratory we have coated anti C3antibody (0.1 g/100 l/well), blocked with 3% BSA and further incubated with optimally diluted (1:150) test sera. FSIgG penicillinase conjugate was used as a probe to detect the filarial specific antigen in bound immunecomplexes. Sera from clinical filarial patients (10 years or more duration) were negative for free circulating antigen but showed high level of immunecomplexed antigen.

IMMUNOFLUORESCENT ASSAY (IFA), Fluorescent tags or labels were first used for histochemical localization of antigen in tissues. This technique is calledimmunofluorescent assay (IFA), a term restricted to qualitative
10

observations involving the use of a fluorescence microscope. In this manner, many types of antigens can be detected either in fixed tissue sections or in live cell suspensions with a high degree of sensitivity and specificity. The presence of a specific antigen is determined by the appearance of localized colour against a dark background. This method is used for rapid identification of microorganisms in cell culture or infected tissue, tumor-specific antigens on neoplastic tissue, transplantation antigens, and CD antigens on T and B cells through the use of cell flow cytometry. Direct Immunofluorescent Assays Fluorescent staining can be categorized as direct or indirect, depending on whether the original antibody has a fluorescent tag attached. In a direct immunofluorescent assay, antibody that is conjugated with a fluorescent tag is added directly to unknown antigen that is fixed to a microscope slide. After incubation and a wash step, the slide is read using a fluorescence microscope. Antigens are typically visualized as bright apple green or orange-yellow objects against a dark background. Direct immunofluorescent assay is best suited to antigen detection in tissue or body fluids, while indirect assays can be used for both antigen and antibody identification. Examples of antigens detected by this method include Legionella pneumophila, Pneumocystis carinii, Chlamydia trachomatis, and respiratory syncytial virus (RSV)

Indirect Immunofluorescent Assays Indirect immunofluorescent assays involve two steps, the first of which is incubation of patient serum with a known antigen attached to a solid phase. The slide is washed, and then an antihuman immunoglobulin containing a fluorescent tag is added. This combines with the first antibody to form a sandwich, which localizes the fluorescence. In this manner, one antibody conjugate can be used for many different types of reactions, eliminating the need for numerous purified, labeled reagent antibodies. Indirect assays result in increased staining, because multiple molecules can bind to each primary molecule, thus making this a more sensitive technique. Such assays are especially useful in antibody identification and have been used to detect treponema, antinuclear, chlamydial, and toxoplasma antibodies, as well as antibodies to such viruses such as herpes simplex, Epstein- Barr, and cytomegalovirus.

11

Fluorescence polarization immunoassay (FPIA), is based on the change in polarization of fluorescent light emitted from a labelled molecule when it is bound by antibody.Incident light directed at the specimen is polarized with a lens or prism so the waves are aligned in one plane. If a molecule is small and rotates quickly enough, the emitted light is unpolarized after it is excited by polarized light. If, however, the labelled molecule is bound to antibody, the molecule is unable to tumble as rapidly, and it emits an increased amount of polarized light. Thus, the degree of polarized light reflects the amount of labelled analyte that is bound. In FPIA, labelled antigens compete with unlabeled antigen in the patient sample for a limited number of antibody binding sites. The more antigen that is present in the patient sample, the less the fluorescence-labelled antigen is bound and the less the polarization that will be detected. Hence, the degree of fluorescence polarization is inversely proportional to concentration of the analyte. This technique is limited to small molecules that tumble freely in solution, usually those analytes with a molecular weight under 2000 d. An additional consideration is nonspecific binding of the labeled conjugate to other proteins in serum. Binding to these molecules would increase polarization, thus falsely decreasing values. FPIA has been used mainly to determine concentrations of therapeutic drugs and hormones. It requires sophisticated instrumentation and is the basis for several automated analyzers on the market today Advantages and Disadvantages of Fluorescent Immunoassay In principle, the use of fluorescence has the potential for high sensitivity and versatility. The methodology is fairly simple, and there is no need to deal with and dispose of hazardous substances. The main problem, however, has been separation of the signal on the label from autofluorescence produced by different organic substances normally present in serum. Another difficulty encountered is the fact that nonspecific binding to substances in serum can cause quenching or diminishing of the signal and change the amount of fluorescence generated. Fluorescence polarization has been developed to overcome some of these problems, and this technique has seen more widespread use. It does, however, require expensive dedicated instrumentation, which may limit its use in smaller laboratories.

12

CHEMILUMINESCENT IMMUNOASSAYS Principle of Chemiluminescence Chemiluminescence is another technique employed to follow antigenantibody combination. It is the emission of light caused by a chemical reaction, typically an oxidation reaction, producing an excited molecule that decays back to its original ground state. A large number of molecules are capable of chemiluminescence, but some of the most common substances used are luminol, acridinium esters, ruthenium derivatives, and nitrophenyl oxalates.When these substances are oxidized, typically using hydrogen peroxide and an enzyme for a catalyst, intermediates are produced that are of a higher energy state. These intermediates spontaneously return to their original state, giving off energy in the form of light. Light emissions range from a rapid flash of light to a more continuous glow that can last for hours. Acridinium esters, for example, emit a quick burst or flash of light, while the light remains for a longer time with luminol and dioxetane. Different types of instrumentation are necessary for each kind of emission. This type of labeling can be used for heterogeneous and homogeneous assays, because labels can be attached to either antigen or antibody. In heterogeneous assays, competitive and sandwich formats are the ones most often used. Smaller analytes such as therapeutic drugs and steroid hormones are measured using competitive assays, while the sandwich format is used for larger analytes such as protein hormones. Advantages and Disadvantages of Chemiluminescent Assays Chemiluminescent assays have an excellent sensitivity, comparable to EIA and RIA, and the reagents are stable and relatively nontoxic. The sensitivity of some assays has been reported to be in the range of attamoles (10-18 mol) tozeptomoles (10-21 mol). Because very little reagent is used, they are also quite inexpensive to perform. The relatively high speed of detection also means a faster turnaround time. Detection systems basically consist of photomultiplier tubes, which are simple and relatively inexpensive. However, false results may be obtained if there is lack of precision in injection of the hydrogen peroxide or if some biological materials such as urine or plasma cause quenching of the light emission. This method has begun to be more widely applied to immunologic testing and has great potential for the future.
13

HYBRIDIZATION TECHNIQUES Spontaneous pairing of complementary DNA strands forms the basis for techniques that are used to detect and characterize genes. Probe technology is typically the basis for identification of individual genes or DNA sequences. A nucleic acid probe is a short strand of DNA or RNA of a known sequence that is used to identify the presence of its complementary DNA/RNA in a patient specimen. Binding of two such complementary strands is known as hybridization and is very specific. The two strands should share at least 16 to 20 consecutive bases of perfect complementarity to form a stable hybrid. Analysis of DNA fragments using probes is typically carried out using a technique known as a Southern hybridization assay, or Southern blot, named after its discoverer, E. M. Southern. After DNA fragments are separated out by gel electrophoresis, the fragments are denatured using alkali and then transferred, or blotted, onto a nitrocellulose or nylon membrane for the hybridization reaction to take place. The transfer is accomplished by placing the membrane on top of the gel and allowing the buffer plus DNA to wick onto the membrane. Traditionally, this procedure takes overnight, but newer methods using vacuum and pressure systems have significantly increased the speed of the transfer. Once the DNA is on the nitrocellulose membrane, heating or UV light is used to cross-link the strands onto the membrane, thus immobilizing them. A labelled probe, whose sequence is complimentary to the sequence of interest, is then added to the membrane for hybridization to take place. The membrane is then washed, and the amount of bound probe remaining is determined by detection of the label. Several nonradiolabeled probes, including enzyme, chemiluminescent, and acridinium labels, have been developed for use, and these appear to be as sensitive as the original radiolabeled probes. The use of these probes avoids the hazards associated with use of radioactivity. The absence of any visible bands indicates an absence of the sequence of interest. APPLICATION: Southern blots are useful to analyze alterations of large spans of DNA, where amplification by the polymerase chain reaction (PCR) may not be practical. Southern blot analysis can reveal polymorphisms in DNA sequence based upon the RFLP profile made visible by the probes. Southern blots have been used to determine the clonal composition of lymphocyte populations. Only when a large number of cloned cells are present do rearranged genes specific for T-cell receptors or immunoglobulins appear in sufficient quantity to produce a detectable band that is different from the normal or germ-line DNA. This is typically an indicator of a lymphoid malignancy such as B-cell lymphoma, chronic myelogenous leukemia, or hairy cell leukemia. Detection of a malignant clone can help distinguish between reactive lymphocytes seen in an inflammatory process and true malignancies. DNA testing is especially helpful in both diagnosis and classification of T-cell malignancies. It has had a tremendous impact on diagnosis of non-Hodgkins lymphomas. Molecular testing can also be used to monitor therapy and remission of lymphomas. If tissue from separate lymphomatous lesions is from the same original source or clone, they usually show identical DNA rearrangements and produce identical bands on a Southern blot. In general, however, Southern blots are complex, timeconsuming to perform, and require a relatively large sample, which can limit its clinical usefulness. Northern blots are performed in a similar manner, but in this technique, RNA is extracted and separated. This tool is used most often to determine the level of expression of a particular messenger RNA species to see if a gene is actually being expressed. Because RNA is a short, single-stranded molecule, it does not have to be digested before electrophoresis, but it has to be
14

denatured to ensure that it is in an unfolded, linear form. Probes are then used to identify the presence of specific bands. In Situ Hybridization In situ hybridization represents a third type of hybridization reaction in which the target nucleic acid is found in intact cells. It provides information about the presence of the tissues. This technique is the same as immunohistochemistry except that nucleic acids instead of antibodies are used as probes. For probes to reach the nucleic acid, they must be small enough, usually limited to 500 bases or less, to penetrate the cells in question.4 Formalin-fixed and paraffin embedded tissue sections are typically used for this procedure. Because preparation of tissue specimens can vary greatly in each lab, it is recommended that an endogenous control probe be used. This probe will react positively with all cells and is used to indicate that penetration of the sample has occurred. Probes have typically been labelled with radioactive substances, but the trend today is toward using fluorescent or enzyme labels. If a fluorescent tag is used, the procedure is called fluorescent in situ hybridization, or FISH. After completion of the procedure, evaluation should be performed by an experienced histopathologist. This technique is used to detect a number of malignancies linked to chromosomal abnormalities, such as chronic myelogenous leukemia, in which there is a translocation from chromosome 9 to chromosome 22, and inherited disorders that occur as a result of chromosomal abnormalities. When FISH is used on metaphase chromosome spreads or interphase nuclei, it can detect numerical alterations or translocation of chromosomal material. DNA Chip Technology Previously, genetic studies examined individual gene expression by northern blotting or examined polymorphisms by gel-based restriction digests and sequence analysis, but with advances in microchips and bead-based array technologies, high-throughput analysis of genetic variation is now possible. Biochips, also called microarrays, are very small devices used to examine DNA, RNA, and other substances. These chips allow thousands of biological reactions to be performed at once. Typically, a biochip consists of a small rectangular solid surface that is made of glass or silicon with Short DNA or RNA probes anchored to the surface. The number of probes on a biochip surface can vary from 10 to 20 up to hundreds of thousands. Usually, the nucleic acid in the sample is amplified before it is analyzed. After amplification, the sample, labelled with a fluorescent tag, is loaded onto the chip. Hybridization occurs on the chips surface, allowing thousands of hybridization reactions to occur at one time. Unbound strands of the target sample are then washed away. The fluorescent-tagged hybridized samples are detected using a fluorescent detector .The intensity of the fluorescent signal at a particular location is proportional to the sequence homology at a particular locus. Complete sequence matches results in bright fluorescence, while single-base mismatches result in a dimmer signal, indicative of a point mutation. Since most single nucleotide polymorphisms (SNPs) are silent and have no apparent functional consequence, the challenge is to identify the set of SNPs that are directly related to or that cause disease. Detection of point mutations can be used for
15

classifying leukemias, molecular staging of tumours, and characterizing microbial agents. One prime example is the determination of genes associated with drug resistance in HIV testing. Identification of such genes guides the physician in selecting a proper drug regimen for a particular patient.

Immunotherapy
Immunotherapy is a form of biologic therapy or biotherapy. It is treatment that uses certain parts of the immune system to fight diseases, including cancer. This can be done in a couple of ways:
16

 Stimulating your own immune system to work harder or smarter Giving you immune system components, such as man-made immune system proteins Immunotherapy is sometimes used by itself to treat cancer, but it is most often used along with or after another type of treatment to boost its effects. For a long time doctors suspected that the immune system had an effect on certain cancers. Even before the immune system was well understood, William Coley, MD, a New York surgeon, first noted that getting an infection after surgery seemed to help some cancer patients. In the late 1800s, he began treating cancer patients by infecting them with certain kinds of bacteria, which came to be known as Coley toxins. Although he had some success, his technique was overshadowed when other forms of cancer treatment, such as radiation therapy, came into use. Doctors have learned a great deal about the immune system since then. This has led to research into how it can be used to combat cancer and exploring many different approaches. In the last few decades immunotherapy has proven useful in treating several types of cancer. The idea of using one's own immune system to fight cancer is tempting, but so far, in most cases immunotherapy hasn't been shown to clearly be better than other forms of treatment. For instance, it seems to work best when treating smaller, early stage cancers, and it may be less helpful for more advanced disease. Its main role at this time is making other forms of treatment better, or giving cancer patients a treatment option that may be less toxic than the usual treatments. But researchers have made important progress in this field. Newer treatments are now being tested that seem to work better and will have a greater impact on the outlook for people with cancer in the future.

Types of immunotherapy
Active immunotherapies stimulate your body's own immune system to fight the disease. Passive immunotherapies use immune system components (such as antibodies) made in the lab. They do not rely on your body to start the attack on the disease. Another way that immunotherapies work is by targeting a certain type of cell. Most of the immunotherapies being used today target one kind of cell or antigen (specific immunotherapies), but there are some that stimulate the immune system in general. These are called non-specific immunotherapies. Sometimes non-specific immunotherapies are used with other treatments to help increase the attack on the cancer. These kinds of treatments are generally only used along with other treatments, so they are called adjuvants. There are other

17

treatments, called targeted therapies that zero in on one type of cell and don't tend to damage other cells. For more information see our document, Targeted Therapy.

Monoclonal antibodies
Monoclonal antibodies are the most widely used form of cancer immunotherapy. Monoclonal antibody therapy uses antibodies that are made in the lab rather than by a person's own immune system. These treatments do not require the person's immune system to start the fight against the cancer. Once the antibodies are given, they can then recruit other parts of the immune system to destroy the cancer cells. The first monoclonal antibodies were made in the lab by fusing a myeloma (a type of bone marrow cancer) cell from a mouse with a mouse B cell that makes a certain antibody. The cell that results from this fusion is called a hybridoma. Combining a B cell that can recognize one special antigen and a long-lived myeloma cell makes the resulting hybridoma cell a long-lasting, antibody-making factory. Because the antibodies made are all identical clones made from a single (mono) hybridoma cell, they are called monoclonal antibodies (sometimes abbreviated as MoAbs or MAbs). The first MAbs were made entirely from mouse cells. One problem with this is that the human immune system will see these antibodies as foreign (because they're from a different species) and then will mount a response against them. This can sometimes cause allergic-type reactions. It also means that the antibodies may only work the first time they are given; after that, the body's immune system is primed to destroy them before they can be helpful. Over time, researchers have learned how to replace some parts of these mouse antibodies proteins with human parts. Depending on how much of the MAb is human, these are called chimeric or humanized antibodies. Some MAbs are now fully human, which means they are likely to be even safer and may be more effective than older MAbs. An even newer approach uses fragments of antibodies instead of whole ones. Smaller pieces may be better able to reach a tumour, which may make them more effective. Clinical trials of monoclonal antibody therapy are also being done on almost every type of cancer. As researchers have found more antigens that are linked to cancer, they have been able to make monoclonal antibodies against more and more cancers. Two types of monoclonal antibodies are used in cancer treatments: Naked monoclonal antibodies are those without any drug or radioactive material attached to them. Conjugated monoclonal antibodies are those joined to a chemotherapy drug, radioactive particle, or a toxin (a substance that poisons cells).
18

Naked monoclonal antibodies

Naked MAbs are the most commonly used MAbs at this time. Although they all work by attaching themselves to specific antigens, they can be helpful in different ways.

Markers for destruction

Some naked MAbs attach to cancer cells to act as a marker for the body's immune system to destroy them. Antibodies now in use in this group include: Rituximab (Rituxan): Rituximab is used to treat B-cell non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and some other diseases. It is a monoclonal antibody against the CD20 antigen, found on B cells. It works, in part, by labelling cells so that the immune system can attack them. Ofatumumab (Arzerra): Ofatumumab is another antibody against the CD20 antigen. It is used mainly to treat chronic lymphocytic leukemia when other treatments are no longer effective. Alemtuzumab (Campath): Alemtuzumab is an antibody against the CD52 antigen, which is found on both B cells and T cells. It is used to treat some patients with B-cell chronic lymphocytic leukemia.

Activation blockers
Some naked MAbs don't really interact with a person's own immune system. Their effects come from their ability to attach to the specific antigens that are working parts of cancer cells or other cells that help cancer cells grow, and stop them from working. These MAbs are also referred to as targeted therapies. Examples of FDA-approved MAbs of this type include: Trastuzumab (Herceptin): Trastuzumab is an antibody against the HER2/neu protein. Large amount of this protein is present on tumor cells in some cancers. When HER2/neu is activated, it helps these cells grow. Trastuzumab stops these proteins from becoming active. It is used to treat breast and stomach cancers that have large amounts of this protein. Cetuximab (Erbitux): Cetuximab is an antibody against the EGFR protein, which is present in large amounts on some tumor cells and helps them grow and divide.
19

Cetuximab blocks the activation of EGFR. It is used to treat some advanced colorectal cancers as well as some head and neck cancers. Panitumumab (Vectibix): This MAb also targets the EGFR antigen. It is used to treat some cases of advanced colorectal cancer.

Side effects of treatment with naked monoclonal antibodies

Monoclonal antibodies are given intravenously (injected into a vein). Compared with the side effects of chemotherapy, the side effects of naked MAbs are usually fairly mild and are often more like an allergic reaction. If they do occur, it is most often while the drug is first being given. Possible side effects can include: Fever Chills Weakness Headache Nausea Vomiting Diarrhoea Low blood pressure Rashes MAbs can also have side effects that are related to the antigens they target. For instance, bevacizumab targets new blood vessel growth, but this can also lead to side effects like bleeding or poor wound healing. MAbs that target EGFR may cause acne-like skin rashes on the face and chest.

Cancer vaccines
Cancer vaccines have been studied for several decades, but advances in this field have been slower than for other forms of immunotherapy. They are still mostly experimental treatments at this time. Most of us know about vaccines given to healthy people to help prevent infections, such as measles and mumps. These vaccines use weakened or killed viruses, bacteria, or other germs to start an immune response in the body. Getting the immune system ready to defend against these germs helps it keep the germs from making people sick. Some so-called "cancer vaccines" are designed to work the same way. For example, new vaccines against the human papilloma virus (HPV) help prevent cervical, vaginal, vulvar, and anal cancer. Vaccines against hepatitis B virus (HBV) may lower some people's risk of getting liver cancer. But these vaccines don't target cancer cells; they target the viruses that can cause these cancers. True cancer vaccines are different from the vaccines that work against viruses. Instead of preventing disease, they are meant to get the immune system to attack a disease that already exists. A true cancer vaccine has cancer cells, parts of cells, or pure antigens. The vaccine increases the immune response against cancer cells that are already in the body. It may be combined with other substances or cells called adjuvants that help boost the immune response even further.
20

Cancer vaccines are thought of as active immunotherapies because they are meant to trigger your own immune system to respond. They are specific because they should only affect the cancer cells. These vaccines don't just boost the immune system in general; they cause the immune system to attack cells with one or more specific antigens. And because the immune system has special cells for memory, it's hoped that the drugs will help keep cancer from coming back. At this time, only one true cancer vaccine has been approved by the FDA. Sipuleucel-T (Provenge) is used to treat advanced prostate cancer. For this vaccine, white blood cells (cells of the immune system) are removed from the patient's blood and exposed to a protein from prostate cancer cells called prostatic acid phosphatase (PAP). These cells are then given back to the patient by infusion into a vein (IV). This process is repeated twice more, 2 weeks apart, so that the patient gets 3 doses of cells. In the body, the cells make other immune system cells attack the patient's prostate cancer. Common side effects include fever, chills, fatigue, back and joint pain, nausea, and headache. A few men may have more severe symptoms, including problems breathing and high blood pressure, which improves with treatment. When used in men with metastatic prostate cancer that no longer responds to hormone therapy, the vaccine helps them live more than 4 months longer on average. Studies to see if this vaccine can help men with less advanced prostate cancer are continuing. Other cancer vaccines have shown some promise in clinical trials, but have yet been approved in the United States to treat cancer. Several types of cancer vaccines are now being studied, with a few reaching late stage clinical trials.

Tumour cell vaccines

Tumour cell vaccines are made from actual cancer cells that have been removed during surgery. The cells are treated in the lab, usually with radiation, so they cannot form more tumours. In most cases, doctors also change the cells in certain ways, often by adding chemicals or new genes, to make them more likely to be seen as foreign by the immune system. The cells are then injected into the patient. The immune system recognizes antigens on these cells, then seeks out and attacks any other cells with these antigens that are still in the body. In some cases, doctors give the vaccine along with substances called adjuvants that increase the immune response. The general boost that adjuvants give to the immune system is meant to make the vaccine work better. Some promising newer versions of these vaccines use tumour cells that are fused to dendritic cells, in the hope of further stimulating the immune system.
21

A possible advantage of tumour cell vaccines over antigen-based vaccines (described in the "Antigen vaccines" section) is that not all cancer antigens have been found yet. Using the whole tumour cell may expose the immune system to a large number of important cancer antigens, including some that researchers have not yet recognized. This may make them more effective. The 2 basic kinds of tumour cell vaccines are autologous and allogeneic. Autologous tumour cell vaccines: Autologous means "coming from the self." An autologous tumour cell vaccine is made from killed tumour cells taken from the same person in whom they will later be used. In other words, cells are taken from you (during surgery), the vaccine is made from them in a lab, and the cells are injected back into you. Autologous cancer cells may be reinjected shortly after surgery, or they may be grown in the lab or frozen and given later. Although autologous tumour cell vaccines are promising, there are some potential drawbacks:

It can be expensive to create a new, unique vaccine for each patient. Cancer cells tend to mutate (change) over time, so an autologous tumour vaccine might become less effective later if the cancer cells in your body change. Depending on the surgery and the size of your tumour(s), you may not have enough

usable cells in the removed tumour to make a vaccine, or there may not be enough for retreatment if the cancer starts growing again. Because of these problems, researchers are also looking at ways to create tumour cell vaccines that could work in any patient with that particular kind of cancer. Allogeneic tumour cell vaccines: Allogeneic means "coming from another." These vaccines use cells of a particular cancer type that originally came from someone other than the patient being treated. Allogeneic vaccines are easier to make than autologous vaccines. They are more like off the- shelf drugs than a vaccine made for just one person. The cells for the vaccine are grown in the lab from a stock of cancer cells kept for that purpose. Some allogeneic tumour vaccines use a mixture of cells that were removed from several patients. The cells are treated and are usually injected along with one or more adjuvant substances to stimulate the immune system. Antigen vaccines Antigen vaccines boost the immune system by using only one antigen (or a few), rather than whole tumour cells that contain many thousands of antigens. The antigens are usually proteins or pieces of proteins called peptides. Antigen vaccines may be specific for a certain type of cancer, but they are not made for a unique patient like autologous cell vaccines are.
22

Scientists have learned how to mass-produce many antigens in the lab. They can also change these antigens to make them more easily recognized by the immune system. This new technology means that large amounts of these very specific antigens can now be given to many patients. Some antigens cause an immune response only in patients with a certain kind of cancer, while others produce immune reactions to more than one kind of cancer. Scientists often combine several antigens in a vaccine to try to get a stronger immune response.

Dendritic cell vaccines

Dendritic cells are special antigen-presenting cells (APCs) that help the immune system recognize cancer cells. They break down cancer cells into smaller pieces (including antigens), then hold out, or "present," these antigens to T cells. This makes it easier for the immune system cells to recognize and attack them. Dendritic cells are the most effective APCs known. Dendritic cell vaccines are autologous vaccines (made from the person in whom they will be used), and must be made individually for each patient. The process used to create them is complex and expensive: Doctors remove some of the cells that grow into dendritic cells (from the blood) and treat them in the lab to make them multiply and turn into dendritic cells. This creates many more dendritic cells than if they just used cells taken from the patient. The dendritic cells are then exposed to cancer cells or cancer antigens. Other methods are to change their genes so that they make their own antigens or to fuse the dendritic cells with tumour cells. These procedures lead to dendritic cells with cancer antigens on their surface. The dendritic cells are then injected back into the patient. The dendritic cells that have cancer antigens on their surface are better able to help the patients immune system recognize and destroy cancer cells that have those antigens on them. The dendritic cell vaccine approach has shown promise in tests in lab animals and in some human studies. It is being studied for use in people with these and other cancers: Prostate cancer, Melanoma, Kidney cancer, colorectal cancer, Lung cancer, Breast cancer, Leukemia, Non-Hodgkin lymphoma. Sipuleucel-T (Provenge), which is approved to treat advanced prostate cancer, is an example of a dendritic cell vaccine. Anti-idiotype vaccines Every B cell or plasma cell makes only one kind of antibody. The unique part of each type of antibody is called an idiotype. Antibodies are made when the immune system responds to
23

antigens. But the immune system also makes some antibodies that treat other antibodies like antigens. In other words, sometimes the body makes antibodies against other antibodies. Scientists believe these antibodies against antibodies are important in helping to keep the immune system in check. Antibodies and antigens fit together like a lock and key. So an antibody to a particular idiotype of another antibody (an anti-idiotype) will usually look like the antigen that triggered cells to make the antibody in the first place (like using the lock itself to make an extra key). Because the anti-idiotype antibodies look like the antigen and appear foreign, injecting them into the body causes the immune system to attack the anti-idiotypes, along with the antigens themselves. Scientists have learned how to make these anti-idiotype antibodies in the lab. They can be used as part of a cancer vaccine because they look like the antigens on the cancer cells in the patient's body. Therefore, they can trigger an immune response against that specific cancer. Researchers consider lymphomas to be the most promising targets for anti-idiotype vaccines. This is because all lymphoma cells have unique antigen receptors not present on normal lymphocytes or other normal cells of the body. These unique antigens can be used to make lymphoma vaccines. Early studies of B-cell lymphoma vaccines have been promising.

DNA vaccines
When tumour cells or antigens are injected into the body as a vaccine, they may cause the desired immune response at first, but they may become less effective over time. This is because the immune system recognizes them as foreign and quickly destroys them. Without any further stimulation, the immune system often returns to its normal (pre-vaccine) state of activity. To get around this, scientists have looked for a way to provide a steady supply of antigens to keep the immune response going. DNA is the substance in cells that contains the genetic code for the proteins that cells make. Cells can be injected with bits of DNA that code for protein antigens. This DNA might be taken up by cells and instruct them to keep making more antigens. These types of therapies are called DNA vaccines. Scientists may be able to do this by removing some of your cells, treating them with DNA that codes for a certain antigen, and then returning them to you. The altered cells would then make the antigen on an ongoing basis to keep the immune response strong. DNA vaccines are now
24

being studied in clinical trials for use against the following cancers, among others: Melanoma, Leukemia, Prostate cancer, Head and neck cancers. Vector-based vaccines These vaccines use special delivery systems (called vectors) to make them more effective. They aren't really a separate category of vaccine; for example, there are vector based antigen vaccines and vector-based DNA vaccines. Vectors are special viruses, bacteria, yeast cells, or other structures that can be used to get antigens or DNA into the body. The vectors are often germs that have been altered to make sure they can no longer cause disease. Vectors may be helpful in making vaccines for a number of reasons. First, they may be used to deliver more than one cancer antigen at a time, which may make the body's immune system more likely to mount a response. Second, vectors such as viruses and bacteria may trigger their own immune responses from the body, which may help make the overall immune response even stronger. Finally, these vaccines may be easier and less expensive to make than some other vaccines. Many clinical trials of vector-based vaccines are now under way.

Immunotherapy for specific cancers

The FDA has approved a number of cancer immunotherapies, including Bacille Calmette Gurin (BCG), interferon-alfa (IFN-alfa), interleukin-2 (IL-2), the sipuleucel-T (Provenge) vaccine, and several monoclonal antibodies. Many other immunotherapies have shown promising results and are moving through the testing process in clinical trials. The cancers listed here are being studied most intensively, but treatments for other cancers are also being looked at. Breast cancer In terms of immunotherapy, only monoclonal antibodies (MAbs) have been approved for use against breast cancer so far. But many other forms of treatment are being studied. Approved The MAb trastuzumab (Herceptin) is used in women with breast cancer whose cancer cells have too many copies of the HER2/neu gene. These genes make extra receptors for growth25

stimulating factors on the cells, which results in a more aggressive form of breast cancer. Trastuzumab attaches to the receptors, blocking the access of the growth factors to the cancer cells and slowing their growth. Other HER2/neu antibodies are now being studied in clinical trials. Bevacizumab (Avastin), a monoclonal antibody that slows blood vessel growth in tumours, has been used along with chemotherapy in some women with advanced breast cancer. Being studied A conjugated MAb, known as trastuzumab-DM1 (or T-DM1) combines the trastuzumab (Herceptin) antibody with a chemo drug. It has shown promise in early studies of women whose breast cancer is no longer responding to trastuzumab alone. Autologous vaccine therapy has lengthened remission and survival times of some women with early breast cancer. This approach is being studied further. A HER2/neu peptide (a small part of the protein made by the HER2/neu gene), used as the antigen in a vaccine, has been shown to cause an increased immune response against the HER2/neu receptor on cancer cells; it is Being studied. Other specific antigen vaccines are also promising. These vaccines are almost always used after primary therapy (lumpectomy and radiation therapy, or mastectomy) and sometimes together with hormonal therapy or chemotherapy, to try to keep the cancer from coming back.

Cervical cancer
Infection with the human papilloma virus (HPV) plays an important role in causing cervical cancer. HPV vaccines are now approved for use to help prevent cervical cancer. Other vaccines that may help treat this cancer are now being tested in clinical trials. Approved Some HPV vaccines are like more traditional vaccines, which work against infections. They are intended to make women immune to some types of HPV, so that when they are exposed to these viruses they will not develop long-term infections. Most cervical cancers may be prevented, if persistent HPV infection is avoided. Two vaccines (Gardasil and Cervarix) can protect against most infections from the 2 types of HPV that cause 70% of cervical cancers. These vaccines are used mainly in girls and young women, although Gardasil is approved for use in boys and young men as well (to help prevent genital warts and other cancers).

Under study

26

Other vaccines are now being studied to help women who already have advanced cervical cancer. These vaccines try to cause an immune reaction to the parts of the virus that contribute to the growth of cervical cancer cells. This may kill the cancer cells or stop them from growing.

Colorectal cancer
Several monoclonal antibodies are now used to treat colorectal cancer. Clinical trials are also using vaccines and many other immunotherapies as adjuvants to surgery, with and without chemotherapy.

Approved
Bevacizumab (Avastin) is a monoclonal antibody against vascular endothelial growth factor (VEGF). By attacking VEGF, the antibody stops tumors from being able to form new blood vessels. It is used along with chemotherapy against advanced colorectal cancer. Cetuximab (Erbitux) is a monoclonal antibody that attacks the epidermal growth factor receptor (EGFR), which normally signals cancer cells to grow and divide. It is used against advanced colorectal cancer, usually along with chemotherapy, in people whose cancer is no longer responding to other treatments. Another monoclonal antibody, panitumumab (Vectibix), also targets EGFR. Unlike cetuximab, this MAb has no parts that come from a mouse, so it may be less likely to cause an allergic reaction when it is given. Panitumumab is used to treat advanced colorectal cancer.

Being studied
A number of autologous and allogeneic tumor cell vaccines has shown early promise, but so far none have improved patient survival time. Some carcinoembryonic antigen (CEA) vaccines have improved the immune response (measured by blood tests) in a large percentage of colorectal cancer patients, but the studies have not been going on long enough to see whether this improves remission or survival times.

Kidney cancer
Immunotherapy has been studied a great deal for advanced kidney cancer, least in part because other treatments like chemotherapy often have not been helpful.

Approved
Two cytokines, IL-2 and IFN-alfa, are treatment options for people with advanced kidney cancer.

27

 Bevacizumab (Avastin), a monoclonal antibody that slows blood vessel growth in tumours, is approved for use against kidney cancer.

Being studied
Whole tumour cell vaccines given along with the adjuvant BCG have shrunk tumours in a small number of people with advanced kidney cancer. Researchers are studying DNA vaccines that insert genes (segments of DNA) into cancer cells, causing the cells to make cytokines. These cytokines help the immune system recognize the cancer cells and also help activate immune system cells to attack those cells. Tumour-infiltrating lymphocytes (TILs) are also being studied to fight kidney cancer. They can be removed from the body and stimulated in the lab by cytokines. When put back into the body, they become more effective than untreated cells from the bloodstream.

Leukemias, lymphomas, and myelomas

Several immunotherapies are now used to treat these blood cell cancers, and many more are being studied.

Approved
Interferon-alfa can be used to treat people with hairy cell leukaemia, chronic myelogenous leukemia, follicular lymphoma, multiple myeloma, and coetaneous (skin) T cell lymphoma. In some cases, interferon is used along with chemotherapy. Denileukin diftitox (Ontak), a combination of IL-2 and diphtheria toxin, is sometimes used to treat cutaneous T cell lymphoma. Rituximab (Rituxan), a monoclonal antibody (MAb), is used to treat some kinds of B cell non-Hodgkin lymphoma and chronic lymphocytic leukemia. Clinical trials are currently testing rituximab against other lymphomas, leukemias, multiple myeloma, and other diseases. Ibritumomab tiuxetan (Zevalin) and tositumomab (Bexxar) are radiolabeled monoclonal antibodies used to treat non-Hodgkin lymphoma, usually in people who aren't helped by other treatments such as chemotherapy or rituximab. They are now being tested to see if they might be helpful earlier in the course of this disease. Alemtuzumab (Campath) is an antibody used to treat B-cell chronic lymphocytic leukaemia (CLL).
28

 Ofatumumab (Arzerra) is an antibody used to treat CLL, usually in people whose leukaemia is no longer responding to other treatments. It is also being studied for use in other types of cancer. Thalidomide (Thalomid) and lenalidomide (Revlimid) are immunomodulating agents that are used to treat multiple myeloma. Brentuximab vedotin (Adcetris or SGN-35) is an antibody that targets the CD30 antigen, attached to a chemo drug called monomethyl auristatin E. It is approved to treat refractory Hodgkin lymphoma and anaplastic large-cell lymphoma.

Being studied
Several other MAbs are being studied in clinical trials for people with leukemia, lymphoma, and multiple myeloma. Anti-idiotype vaccines have shown promising results in clinical trials against B-cell nonHodgkin lymphomas, but are not yet FDA approved.

Lung cancer
Better treatments are needed for lung cancer, especially for advanced disease. Immunotherapy may help people live longer without the severe side effects sometimes seen with chemotherapy. Thus far, only monoclonal antibodies have been approved for use against lung cancer, although many other forms of immunotherapy are being studied.

Approved
The monoclonal antibody bevacizumab (Avastin) slows the growth of tumour blood vessels by targeting the VEGF protein. For some patients with non-small cell lung cancer (NSCLC), adding it to standard chemotherapy may help them live longer than chemotherapy alone.

Being studied
Stimuvax (BLP25) is a peptide (antigen) vaccine that is encased in a fat droplet (liposome) to make it work better. A small study of patients with advanced NSCLC suggested it might improve survival time. Larger studies are needed to confirm this.

Melanoma
29

Melanoma is probably the most-studied cancer when it comes to immunotherapy. Part of this is because doctors think this cancer may be more vulnerable to immune system responses. In rare cases, these cancers have been seen to shrink or even disappear without treatment. It is thought that this may be because of an effective immune response by the body. Another reason immunotherapy has been studied more in melanoma is because other treatments, like chemotherapy, don't work as well against this cancer as they do for most cancers.

Approved
The cytokines IFN-alfa and IL-2 are approved to treat people with metastatic melanoma. Ipilimumab (Yervoy) is approved to treat advanced melanoma.

Ovarian cancer
Immunotherapy is not used routinely to treat ovarian cancer. Several types of immunotherapy, including cancer vaccines and MAbs, are now being studied. The monoclonal antibody bevacizumab (Avastin) slows the growth of tumor blood vessels by targeting the VEGF protein. It can slow the growth of advanced ovarian cancer, although it's not yet clear if it helps women live longer. Injection of interleukin-2 (IL-2) directly into the peritoneal cavity (the part of the belly that contains the ovaries, uterus, and digestive organs) of women with recurrent ovarian cancer is being studied. Early studies suggest the treatment may increase the length of remissions (periods of time with no signs of cancer) after surgery. Placing tumour-infiltrating lymphocytes (TILs) along with interleukin-2 directly into the peritoneal cavity have also shown promise and are being studied. A monoclonal antibody that attaches to certain antigens on both ovarian cancer cells and to certain spots on T cells (a bi-specific antibody) has shown promise when used with IL-2. The antibody causes T cells to bind to and attack the cancer cells. Early studies have shown that radiolabeled MAbs against ovarian cancer may help more women live longer. Several forms of antigen vaccines are being studied to treat ovarian cancer.

Prostate cancer
Immunotherapy has not been a routine part of treating prostate cancer treatment. This may change with the approval of sipuleucel-T (Provenge). Most other prostate cancer immunotherapies now being studied are vaccines. They are designed to cause immune

30

responses to antigens present only on prostate cells, such as prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA).

Approved
For sipuleucel-T (Provenge), white blood cells are removed from a patient's bloodstream and treated in the lab with a prostate cancer antigen and other substances to become dendritic cells. When put back in to the patient, the dendritic cells can show this antigen to other immune system cells, which are then better able to recognize and attack the cancer cells. When used to treat men with hormonerefractory prostate cancer, this vaccine helped them live longer.

Being studied
GVAX is an autologous whole cell vaccine. It is made by removing cancer cells from the patient during surgery and modifying them in the lab to express GM-CSF (to help stimulate the immune system). The cells are irradiated so they can't grow any more. They are then injected back into the patient to cause an immune response. Early studies of patients with advanced prostate cancer that no longer responded to hormone therapy have shown some promising results in terms of survival time. This vaccine is now being tested against the current standard chemotherapy regimen for prostate cancer. Researchers also are looking into using a part of the prostate-specific antigen (called a peptide) as the basis of a vaccine. DNA vaccines, monoclonal antibodies, and cytokines have also shown promise and are being tested in clinical trials.

REFERENCES Ashihara, Y, Kasahara, Y, and Nakamura, RM. (2007), Immunoassays and immunochemistry. In McPherson, RA, and Pincus, MR (eds): Henrys Clinical Diagnosis and Management by Laboratory Methods, ed. 21. Saunders Elsevier, Philadelphia, pp. 793818 Ault KA, (2007). Effect of prophylactic human papillomavirus L1 viruslike- particle vaccine on risk of cervical intraepithelial neoplasia grade 2, grade 3, and adenocarcinoma in situ: a combined analysis of four randomised clinical trials. Lancet; 369:18611868 Carpenter, AB (2007). Immunoassays for the diagnosis of infectious diseases. In Murray (eds): Manual of Clinical Microbiology, ed. 9. ASM Press, Washington, DC pp. 257269 Chen R, Gopal AK, Smith SE, (2010). Results of a pivotal phase 2 study of brentuximab vedotin (SGN-35) in patients with relapsed or refractory Hodgkin lymphoma. Blood
31

(ASH Annual Meeting Abstracts); 116:283. Collins, AB, Colvin, RB, Nousari, CH, and Anhalt, GJ,( 2006). Immunofluorescence methods in the diagnosis of renal and skin diseases. In Detrick, B, Hamilton, RG, and Folds, JD (eds): Manual of Molecular and Clinical Laboratory Immunology, ed. 7. ASM Press, Washington, DC, pp. 414423. Consortium for Retrovirus Serology Standardizations. Serologic diagnosis of human immunodeficiency virus infection by Western blots testing. JAMA 260:674679, 1988 Fleischer, DM, and Wood, RA (2006) Tests for immunological reactions to foods. In Detrick, B, Hamilton, RG, and Folds, JD (eds): Manual of Molecular and Clinical Laboratory Immunology, ed. 7. ASM Press, Washington, DC, pp. 975973. Forbes, BA, Sahm, DF, and Weissfeld, AS. (2007) Bailey and Scotts Diagnostic Microbiology, ed. 12. Mosby Elsevier, St. Louis pp. 147158. Homburger, HA. (2007) Allergic diseases. In McPherson, RA, and Pincus, MR (eds): Henrys Clinical Diagnosis and Management by Laboratory Methods, ed. 21. Saunders Elsevier, Philadelphia, pp. 962971. Jandreski, MA. (1998). Chemiluminescence technology in immunoassays. Lab Med 29:555560, Kim, R, Emi, M, and Tanabe, K., (2007) Cancer immunoediting from immune surveillance to immune escape. Immunology 121: 114. Kricka, LJ. (2008). Principles of immunochemical techniques. In Burtis, CA, Ashwood, ER, and Bruns, DE (eds): Tietz Fundamentals of Clinical Chemistry, ed. 6. Saunders Elsevier, St. Louis, pp. 155170 Lindsley, MD, Warnock, DW, and Morrison, CJ. (2006), Serological and molecular diagnosis of fungal infections. In Detrick, B, Hamilton, RG, and Folds, JD (eds): Manual of Molecular and Clinical Laboratory Immunology, ed. 7. ASM Press, Washington, DC, pp. 569605. National Academy of Clinical Biochemistry (2005). Laboratory Medicine Practice Guidelines: Tumor Markers. Draft Guidelines 2005. Www.nacb.org. Accessed July 2012 Remaley, AT, and Hortin, GL. (2006) Protein analysis for diagnostic applications. In Detrick, B, Hamilton, RG, and Folds, JD (eds): Manual of Molecular and Clinical Laboratory Immunology, ed. 7. ASM Press, Washington, DC, pp. 721. Restifo NP, Robbins PF, Rosenberg SA (2008). Principles of immunotherapy. In: DeVita VT, Hellman S, Rosenberg SA, eds. Cancer: Principles and Practice of Oncology. 8th ed. Philadelphia, Pa: Lippincott Williams & Wilkins: 351368. Roitt, I, Brostoff, J, and Male, D. (2001) Immunology, ed. 6. Mosby, London, England, Sable, C. (2008). Advances in antifungal therapy. Ann Rev Med 59:361,
32

Salgaller ML. (2000) Immune adjuvants. In: Rosenberg SA, ed. Principles and Practice of the Biologic Therapy of Cancer. 3rd ed. Philadelphia, Pa: Lippincott Williams & Wilkins 584601 Shea, YR. (2007) Algorithms for detection and identification of fungi. In Murray, PR et al. (eds): Manual of Clinical Microbiology, ed. 9. ASM Press, Washington, DC, pp. 257269. Tuma, R. (2007) Inconsistency of HER2 raises questions. J Natl Cancer Inst 99(14):1064 1065, Von Muhlen, CA, and Nakamura, RM. (2007) Clinical and laboratory evaluation of rheumatic diseases. In McPherson, RA, and Pincus, MR (eds): Henrys Clinical Diagnosis and Management by Laboratory Methods, ed. 21. Saunders Elsevier, Philadelphia, pp. 916932.

33