Biomarkers in Clinical Medicine Xiao-He Chen, Shuwen Huang, and David Kerr

unit 4. integration of biomarkers into epidemiology study designs

chapter 17. Biomarkers in clinical medicine Xiao-He Chen, Shuwen Huang, and David Kerr

Summary Introduction

Biomarkers have been used in disease severity, and 5) prognostic Health sciences have been clinical medicine for decades. With biomarkers to predict future disease experiencing a shift from population- the rise of genomics and other course, including recurrence, based approaches to individualized advances in molecular biology, response to therapy, and monitoring practice. The focus on individuals biomarker studies have entered a efficacy of therapy (1). Biomarkers could make public health strategies whole new era and hold promise can indicate a variety of health or more effective by allowing for early diagnosis and effective disease characteristics, including practitioners to direct resources treatment of many diseases. A the level or type of exposure to towards those with the greatest biomarker is a characteristic that is an environmental factor, genetic need. However, the success of objectively measured and evaluated susceptibility, genetic responses to these efforts will largely depend Unit 4 as an indicator of normal biological environmental exposures, markers on the continued identification processes, pathogenic processes of subclinical or clinical disease, or of biomarkers that reflect the or pharmacologic responses to a indicators of response to therapy. individual’s health status and risk 17 Chapter therapeutic intervention (1). They This chapter will focus on how at key time points, and successful can be classified into five categories these biomarkers have been used integration of these biomarkers into based on their application in different in preventive medicine, diagnostics, medical practice. To be clinically disease stages: 1) antecedent therapeutics and prognostics, useful, tests for biomarkers must biomarkers to identify the risk of as well as public health and their have high predictive accuracy, developing an illness, 2) screening current status in clinical practice. and be easily measurable and biomarkers to screen for subclinical reproducible, minimally invasive, disease, 3) diagnostic biomarkers and acceptable to patients and to recognize overt disease, 4) physicians (2). Once a proposed staging biomarkers to categorise biomarker has been validated, it

Unit 4 • Chapter 17. Biomarkers in clinical medicine 303 can be used to assess disease risk level has been used as a marker in of the TYR gene that encodes a in a general population, confirm investigating adolescents’ cigarette R402Q amino acid substitution, diagnosis of disease in an individual consumption. A biomarker of effect previously shown to affect eye patient, and tailor an individual’s is a measurable alteration of an colour and tanning response, was treatment (choice of drug treatment endogenous factor that is shown associated with increased risk of or administration regimes). In to be linked with impairment or developing cutaneous melanoma evaluating potential drug therapies, disease resulting from exposure to (CM) and basal cell carcinoma a biomarker may be used as a an exogenous agent. For example, (BCC); 2) variations in a haplotype surrogate for a natural endpoint, such the alteration in pulmonary function (set of closely associated genes) as survival or irreversible morbidity. tests in children after exposure to near the ASIP gene, known to If a treatment alters the biomarker, environmental tobacco smoke is affect pigmentation traits, conferred which has a direct connection to a biomarker of effect (3). Somatic significant risk of CM and BCC; and improved health, the biomarker mutations have been used as 3) an eye colour variant in gene serves as a surrogate endpoint for biomarkers of effect after exposure TYRP1 was also associated with evaluating clinical benefit. More to carcinogens. A biomarker of risk of CM (5–7). The relationship recently, with rapid advances susceptibility indicates individual between these biomarkers and their in the molecular approaches to factors that can affect response to relationship with clinical medicine biology, genetics, biochemistry and environmental agents. These reflect are illustrated in Figure 17.1. medicine, and in particular with the variations between individuals There are two layers of exposure rise of genomics, transcriptomics, in genetic structure, some of and effect biomarkers. The first proteomics and metabolomics, which make the individual more represents hazardous exposures molecular biomarkers appear to susceptible to health effects from to a healthy human body that could hold the promise of transforming environmental exposures (4). For cause negative biological effects medical practice into personalized example, skin cancer is related to (e.g. functional changes, somatic medicine—the right treatment at the excessive sun exposure, but not mutations) and eventually cause right dose for the right person at the everyone develops skin cancer even disease. Another layer indicates right time for the right outcome. with the same amount of exposure. treatment exposures to a diseased Three recent studies revealed human body that could induce Context and public health that genetic variants associated positive biological effects and lead significance with three sections of genes were to the improvement of conditions found to be linked with increased or to the complete recovery from Clinical medicine covers disease risk of skin cancer: 1) the variant disease. Susceptibility biomarkers prevention, diagnosis and treatment. Biomarkers play a critical Figure 17.1. Simplified flowchart of classes of biomarkers (indicated by boxes) role in all these aspects. There are representing a continuum of changes. Solid arrows indicate progression, if it occurs, to the next class of marker. Dashed arrows indicate that individual biomarker three major types of biomarkers: influences and/or indicates the rates of progression biomarkers of exposure, effect and susceptibility. A biomarker of exposure is an exogenous chemical or its metabolite(s), or the product of an interaction between a xenobiotic agent and some target molecule or cell that is measured in a compartment within an organism. Specific markers of exposure include the presence of a xenobiotic compound or its metabolites in body tissues or fluids and in excretory products. For example, blood lead concentration has been used as a marker for lead exposure; saliva cotinine (a metabolite of nicotine) Source: Adapted from (106)

304 are present in every step of the preventive medicine for several and activating mutation in Ras process. For example, some decades to: screen before birth for oncogene and loss of function individuals exposed to air pollutants genetic disorders, such as Down mutations in APC and p53 tumour show severe biological effects and syndrome; screen newborn babies suppressor genes for colon cancer) manifest disease symptoms, while for genetic diseases, such as (12). Subtypes of these cancers and others experience little or no effect. phenylketonuria (PKU) (9); check most other common diseases are The same discrepancy appears with whether an individual is a carrier less deterministic; even apparently drug treatment. While some patients for a recessive disorder (where simple Mendelian disorders may benefit and are cured, others show abnormal genes must be inherited prove to have widely variable no effect from treatment or develop from both parents to lead to the clinical phenotypes. For example, severe side-effects or die. In clinical condition), such as cystic fibrosis; thalassaemia, an apparently simple medicine, the first layer is more and indicate whether someone genetic disease, has substantial related to disease prevention and with a family history of a late-onset complexities (13). Individuals with diagnosis, while the second layer is disease, such as Huntington’s, exactly the same globin mutations more relevant to disease treatment is likely to develop the disease. may suffer either from a severe life- and recovery. These tests are aimed largely at threatening disorder or be relatively single-gene disorders that have unaffected. Despite this, great Biomarkers in preventive Mendelian patterns of inheritance. efforts have been made towards medicine The identification of genetic variants simultaneous, systematic analysis responsible for diseas, in these of larger numbers of biomarkers Preventive medicine aims to promote single-gene disorders can lead for disease prediction, although and preserve health and longevity directly to clinically helpful and these approaches are more suited in individuals and populations, use reasonably accurate predictions to research than routine diagnostic epidemiological approaches to and diagnosis of disease. Early activity. Biomarkers may help predict define high-risk groups, prevent and diagnosis and proper treatment those individuals more susceptible to limit disease and injury, facilitate can make the difference between common disorders, so that specific early diagnosis through screening lifelong impairment and healthy attention can be directed towards and education, enhance quality of development. them (e.g. enrolment in a screening the health care system and improve Common, complex diseases programme). However, translating quality of life. To realize these aims, such as cancer, heart disease and these biomarkers into clinical medical practitioners need the diabetes contribute to the major medicine to help prevent people proper tools to facilitate decision- disease burden and mortality both in having these common diseases still making and effect evaluation; developed and developing countries. has a long way to go. As Kofi Annan, biomarkers play an important role in These common diseases are caused the former Secretary General of these goals. by genetic and environmental the United Nations, said, “We are Exposure biomarkers have been factors (e.g. lifestyle and diet, and under no illusion that preventive used in the workplace for many the interaction between them). strategies will be easy to implement. years to identify exposed individuals. Therefore, it is very difficult to define For a start, the costs of prevention For example, macromolecule a single biomarker that could identify have to be paid in the present, while Unit 4 adducts and mutagenicity in urine the risk of developing a particular its benefits lie in the distant future. have been successfully applied disease. Although there are some And the benefits are not tangible— to identify workers exposed to rare subtypes of common diseases, when prevention succeeds, nothing 17 Chapter carcinogens and as indicators of such as breast and colorectal happens. Taking such a political risk changes of exposure. Biomarkers cancer, with a clear hereditary when there are few obvious rewards of renal effects of cadmium, lead pattern, biomarkers for a single requires conviction and considerable effects on haemoglobin synthesis or several defective genes could vision.” (14). and organophosphate effects on indicate a lifetime risk of developing cholinesterase activities have been these cancers (e.g. overexpression Biomarkers in diagnostics validated and are widely used in of HER2/neu oncogene and loss routine monitoring activities (8). of function mutations in BRCA1 Biomarkers have been used in Antecedent and screening and BRCA2 tumour suppressor disease diagnosis for over a century, biomarkers have been used in genes for breast cancer (10–12), beginning when the ABO blood

Unit 4 • Chapter 17. Biomarkers in clinical medicine 305 group system was first discovered the early detection and diagnosis microdeletion involving the proximal and used to detect ABO haemolytic of chromosome and single-gene part of the long arm of chromosome disease of the newborn (HDN) and disorders. Both cytogenetic and 15q (15q11–12). It is now known transfusion reactions. The four molecular genetic biomarkers have that if the deletion occurs de novo basic ABO phenotypes are O, A, B been used to accomplish this. on the paternally inherited number and AB. After it was found that blood Conditions caused by a change 15 chromosome, the child will have group A’s red blood cells (RBCs) in the number (e.g. aneuploidy) or Prader-Willi syndrome; a deletion reacted differently to a particular structure of chromosomes (e.g. occurring at the same region on antibody (later called anti-A1), the translocation, inversion, deletion, the maternally inherited number blood group was divided into two and duplication) are known as 15 chromosome causes Angelman phenotypes, A1 and A2. RBCs with chromosome disorders. Biomarkers syndrome. Non-deletion cases also the A1 phenotype react with anti-A1 used in the chromosome analysis exist and are often due to uniparental and account for about 80% of blood developed in 1956 soon led to the disomy (i.e. both homologues of type A. RBCs with the A2 phenotype discovery that several previously a chromosome pair are inherited do not react with anti-A1 and described conditions were due to an from only one of the parents), with makeup about 20% of blood type A. abnormality in chromosome number. both number 15 chromosomes HDN, caused by ABO antibodies, For example, in Turner syndrome, being paternal in origin in Angelman occurs almost exclusively in infants only one intact X chromosome is syndrome and maternal in origin in of blood group A or B who are present (45, X); all or part of the Prader-Willi syndrome. This “parent born to group O mothers (15). This second X is deleted. Patients with of origin” effect is referred to as is because the anti-A and anti-B Down syndrome have an extra genomic imprinting and methylation formed in group O individuals tends chromosome 21 (47, XX/XY, +21). of DNA. Here, CNV and mythelation to be of the IgG type (and therefore Patau syndrome is the result of biomarkers, coupled with clinical can cross the placenta), whereas trisomy 13, while trisomy 18 causes observations, have helped identify the anti-A and anti-B found in the Edwards syndrome. The biomarker new underlying genetic mechanisms serum of group B and A individuals, test in this case is assessment of (18). respectively, tends to be of the IgM chromosome numbers. The most widely used type. Although uncommon, cases of Microdeletion/microduplication biomarkers identified during the last HDN have been reported in infants syndromes are a group of few decades are for the diagnosis born to mothers with blood group chromosome disorders that could be of single-gene disorders. More A2 (16) and blood group B (17). detected by biomarker copy number than 10 000 human diseases are The most common cause of death variation (CNV). “Micro” represents believed to be caused by defects in from a blood transfusion is clerical submicroscopic, meaning that these single genes, affecting 1–2% of the error, in which an incompatible deletions, normally smaller than population (18). The disease can be type of ABO blood is transfused. 3Mb, cannot be detected using a relatively trivial in its effects (e.g. If a recipient who has blood group microscope. New technologies, colour blindness), or lethal like Tay- O is transfused with non-group especially array comparative Sachs (a fatal inherited disease of O RBCs, the naturally occurring genomic hybridization (array-CGH), the central nervous system; babies anti-A and anti-B in the recipient's enabled many malformations with Tay-Sachs lack an enzyme serum binds to their corresponding and syndromes to be recognized. called hexosaminidase A (hex A) antigens on the transfused RBCs. Figure 17.2 shows recently detected which is necessary for breaking These antibodies fix complement or confirmed microdeletions/ down certain fatty substances and cause rapid intravascular duplications collected in DECIPHER in brain and nerve cells). Other haemolysis, triggering an acute (DatabasE of Chromosomal disorders, though harmful to those haemolytic transfusion reaction Imbalance and Phenotype in afflicted with them, appear to offer that can cause disseminated Humans using Ensembl Resources) some advantage to carriers. For intravascular coagulation, shock, (https://decipher.sanger.ac.uk/). example, carriers of sickle cell acute renal failure and death. Applications of new biomarkers in anaemia and thalassemia appear Routine biomarker tests can confirm these disorders have generated to have enhanced resistance to the diagnosis. particular interest. For example, malaria. Some other examples Another important use of most Angelman and Prader- of single-gene diseases are biomarkers in clinical medicine is Willi syndromes are related to cystic fibrosis, Marfan syndrome,

306 Figure 17.2. Known syndrome microdeletions/duplications from DECIPHER available in clinical DNA diagnostic (https://decipher.sanger.ac.uk/) laboratories (19). To date, approximately 20 000 chromosomal abnormalities have been registered on laboratory databases, and over 10 000 single-gene traits and disorders have been identified. While on an individual basis most of these are very rare, collectively they make a major contribution to human morbidity and mortality. Screening biomarkers have been used to help early detection and diagnosis of these disorders. Current nationally managed screening programmes in the United Kingdom include: antenatal screening for Down syndrome, sickle-cell disease and thalassemia; newborn screening for PKU, congenital hypothyroidism, sickle- cell disease, thalassemia, cystic fibrosis and hearing impairment; and adult screening for breast cancer, cervical cancer and sight- threatening diabetic retinopathy. Applications of biomarkers in these screening programmes have greatly helped people with early intervention (e.g. diet), decision-making (e.g. marriage and childbearing) and Huntington disease and hereditary gene disorder where molecular early treatment. haemochromatosis. Early detection biomarkers play an important role in Many of our most common and diagnosis of these disorders rely diagnosis. Cystic fibrosis is the most diseases run in families, but they on rapidly developed biomarkers, common life-threatening autosomal lack the simple inheritance patterns especially molecular biomarkers. recessive disorder in Caucasians, characteristic of single-gene For example, phenylketonuria (PKU) affecting 1 in 2500 newborns. The disorders. In complex disorders, is a common genetic disorder (1 in disease results from a defect in pedigrees reveal no Mendelian 12 000 births) which results from the cystic fibrosis transmembrane inheritance patterns, and gene Unit 4 a deficient enzyme required for conductance regulator (CFTR). mutations are often neither the metabolism of the amino acid Diagnosis of cystic fibrosis is based sufficient nor necessary to explain phenylalanine. Failure to recognize on the combination of suggestive the disease phenotype (20). The 17 Chapter the disorder early in life results clinical symptoms, elevated chloride more complicated nature makes in mental retardation. Routine levels on a standardized sweat genetic analysis for early diagnosis biochemical screening of newborn test, family history and genetic much more difficult than for single- infants for PKU was recommended testing to identify the presence of gene or chromosomal disorders. by the Ministry of Health in the United CFTR mutations. Over 1000 CFTR There has been significant activity Kingdom in 1969, after it had been mutations have been identified, with in the development of biomarkers shown that a low phenylketonuria the ΔF508 being the most common for the diagnosis and prognosis diet could prevent the severe in all ethnic groups. Mutation of common diseases such as learning disabilities. Cystic fibrosis panels using PCR to detect the heart disease and cancer. Since is another example of a single- most common mutations are widely the start of the 21st century, there

Unit 4 • Chapter 17. Biomarkers in clinical medicine 307 has been an explosion of high- 69% specificity, and is only useful receiving standard chemotherapy throughput genomic, proteomic in monitoring therapy for advanced (35). Over 90% of patients receiving and metabolomic technologies that breast cancer or recurrence (33). imatinib respond positively to initial have furthered our understanding For a biomarker to be valuable in treatment, and many experience of molecular mechanisms that clinical practice, it must have high complete remission. The presence underlie these common diseases. sensitivity and specificity. or absence of this biomarker These technologies are forming the The lack of sensitivity and can also facilitate appropriate basis for more advanced molecular specificity for single markers is application of another drug, diagnostics in which DNA, RNA, not surprising given the degree of Busulfan (Busilvex®, Myleran®). protein and metabolite data are heterogeneity present in both solid According to the prescribing integrated with traditional clinical tumours and the human population at information, “Busulfan is clearly less profiles to give clinicians the ability to large. Thus, a prevailing hypothesis effective in patients with chronic accurately diagnose diseases (21– is that a panel of biomarkers would myelogenous leukemia who lack the 24). To date, although a large number cumulatively possess a higher Philadelphia (Ph1) chromosome. of candidate biomarkers have been sensitivity and specificity than any Also, the so-called ‘juvenile’ type identified which have the potential single biomarker (34). of chronic myelogenous leukemia, to be used for both early diagnosis typically occurring in young children and therapeutic guidance in cancer, Biomarkers in therapeutics and associated with the absence heart disease, Alzheimer's disease, and prognostics of a Philadelphia chromosome, rheumatoid arthritis and asthma, responds poorly to Busulfan. The only a fraction have been approved Biomarkers play an important role drug is of no benefit in patients in tests by the US Food and Drug in disease treatment, prognosis whose chronic myelogenous Administration (FDA) (Table 17.1) and management in many different leukemia has entered a ‘blastic’ (25). Even among those biomarkers ways. Several common diseases phase.” Therefore, the presence of approved by FDA tests, few of them are very heterogeneous, as the the Philadelphia chromosome can have lived up to their initial promise. same disease may show different aid the diagnosis and treatment of For example, the carcinoembryonic phenotypes, may be caused by these diseases. antigen (CEA) test for colon cancer different genetic mechanisms, and Biomarkers can also work as initially claimed to have 100% may respond differently to the same a surrogate endpoint to indicate sensitivity and specificity (26); later treatment. For example, biomarker the treatment response. A clinical investigations found much lower Philadelphia chromosome, t(9;22) endpoint is a characteristic or levels of discrimination (27). The translocation, is found in 95% of variable that reflects how a patient cancer antigen 125 (CA125) test cases of chronic myeloid leukaemia feels, functions or survives. A was also hailed as a groundbreaking (CML) and in some cases of acute surrogate endpoint is a biomarker diagnostic test for ovarian cancer, lymphoblastic leukaemia (ALL). intended to substitute for a clinical but it is now considered less CML is caused by a chromosomal endpoint. It is expected to predict reliable, as only 50% of women rearrangement that creates a clinical outcome (benefit or harm, with treatable, early-stage ovarian fusion between two normal proteins or lack of benefit or harm) based cancer have increased CA125 levels, producing one abnormal protein, on epidemiological, therapeutic, while women with other conditions, BCR-ABL, that promotes a rapid pathophysiological or other such as endometriosis, can have increase in the number of white scientific evidence. For example, in increased levels (28). Prostate blood cells. This biomarker led to the cancer treatment, the application of specific antigen (PSA), currently development of Gleevec® (imatinib FDG-PET, which is on clinical trial the best overall serum biomarker mesylate), which binds specifically by the US FDA, National Cancer for prostate cancer, has high to BCR-ABL and inhibits its action. Institute (NCI) and the Center for sensitivity (greater than 90%) but Appropriate prescription of the drug Medicare Studies (CMS), may have low specificity (~25%), which results can be confirmed by a diagnostic an important role as a surrogate in many men having biopsies when test that detects the presence of the endpoint for assessing the clinical they do not have detectable prostate BCR-ABL gene. Studies show vastly efficacy of oncologic therapies. FDG- cancer (29–32). The serum tumour improved response rates and lower PET represents fluorodeoxyglucose biomarker for breast cancer (CA15– toxicity for CML patients receiving positron emission tomography, an 3) has only 23% sensitivity and imatinib compared with patients imaging method to detect gamma

308 Table 17.1. US FDA-published list of valid genomic biomarkers, approved drug labels, and test recommendation (1 = test required, 2 = test recommended, 3 = information only) Unit 4 Chapter 17 Chapter

Source: (25)

Unit 4 • Chapter 17. Biomarkers in clinical medicine 309 rays. It measures glucose uptake 40). These genetic variations could by 10–15% to keep the systemic by tumours using a radioactive be used as biomarkers to direct a level of the drug comparable to form of fluorine incorporated in physician’s drug choice for a patient that in patients with normal TPMT a sugar molecule. Tissues that and prevent adverse drug reactions. who have been given a standard accumulate radioactive glucose are For example, the anticoagulant drug dose of the drug (http://www. visible through positron emission warfarin, marketed as Coumadin® personalizedmedicinecoalition.org/ tomography. It is believed that by Bristol-Myers Squibb, is used communications/pmc_pub_11_06. FDG-PET could become a tool for to prevent potentially fatal clots in php). (More information on genomic gauging a cancer patient’s response blood vessels. Approximately 2 biomarkers for drug usage can be to chemotherapy or radiation by million people start warfarin therapy found at http://www.fda.gov/Drugs/ accurately measuring tumour each year to prevent blood clots, ScienceResearch/ResearchAreas/ metabolism. Physicians will quickly heart attacks and stroke. However, Pharmacogenetics/ucm083378. know whether or not the tumour is too much or too little of the drug htm.) responding to therapy and when to can cause serious, life-threatening switch therapies to provide the best bleeding or blood clots. According to Biomarkers in clinical trials chance for curing or managing the the FDA’s adverse events reporting and drug discovery cancer. Cervical tumour uptake database, complications from of F-18 FDG, measured as the warfarin are the second (just after With rapid advances in the molecular maximal standardized uptake value that from insulin) most common approaches to biology, genetics,

(SUVmax) by PET, and its association reason for patients to go to the biochemistry and medicine, a with treatment response and emergency department. Variability significant number of new drugs and prognosis in patients with cervical in the response to warfarin has treatments have been developed. cancer were evaluated. It was found been linked to mutations in two But most of these discoveries that a higher SUVmax was associated genes: CYP2C9 and VKORC1. still remain in the research field. with an increased risk of lymph node Clinical studies have shown that Efficiently and effectively translating metastasis at diagnosis (P = 0.0027) patients with variations in these two these discoveries into clinical (36). genes may need a lower warfarin practice is complex and involves Biomarkers can help reduce dose than patients without those the integration of scientific rationale adverse drug reactions. Studies variations. Recently, the FDA and the regulatory process. estimate that over 2 million serious cleared the first test to detect gene Various models depict translational adverse drug reactions (ADRs) variants in patients that are sensitive research as a process occurring occur annually in the United to the anticoagulant warfarin. in two stages (42–44). The first States, and as many as 137 000 Thiopurine methyltransferase (sometimes referred to as type 1 deaths are caused by ADRs (37). (TPMT) is another example of a (T1) translation) uses the findings Using biomarkers to indicate if the biomarker being applied to drug from basic research, including patient is suitable for treatment treatment. TPMT is responsible preclinical studies, to inform the with certain drugs, and what dose for inactivating purine drugs used development and testing of an is appropriate for the patient, could for treating acute lymphoblastic intervention in clinical trials, such prevent some of these deaths. Any leukaemia (ALL) and other diseases as Phase I-III clinical trials. The given drug can be therapeutic to (41). Variations in the TPMT gene second (type 2 (T2) translation) some individuals and ineffective can cause changes in TPMT involves the translation of findings to others, and likewise some enzymatic activity and thus drug from clinical research into clinical individuals suffer from adverse drug metabolism. One in 300 patients has and public health practice and policy effects whereas others experience TPMT deficiency. In these patients, (42,43). This section discusses drug resistance. Often, distinct the normal dose of purine causes an how biomarkers have been used in molecular mechanisms underlie accumulation of active compound, clinical trial and drug development, therapeutic and adverse effects. which may lead to a potentially fatal and what changes can be brought Studies have linked differences in bone marrow reaction resulting in about by biomarker application in drug responses to differences in leucopenia, an abnormal lowering these fields in clinical medicine. genes that code for the production of the white blood cell counts. A clinical trial is defined as a of drug-metabolizing enzymes, drug Therefore, if TPMT deficiency prospective study comparing the transporters or drug targets (38– is detected, the dose is lowered effect and value of intervention(s)

310 against a control in human beings (imatinib mesylate) specifically involve appropriate animal models. (45). Clinical trials are commonly targets this enzyme by attaching to In cancer studies, for example, the classified into four phases. Phase the cancerous cells and stopping complexities of modeling human I trials select drug dose, schedule them from growing and spreading. cancer in experimental systems and associated toxicities. Phase But in some patients, the cancer are well known and have impeded II trials determine the degree of cells mutate just enough to be cancer drug development over the efficacy and govern admission to resistant to imatinib. Bristol-Myers years (46). Genetically engineered Phase III testing. Phase III trials Squibb produced another drug, cancer models have improved compare a new treatment against dasatinib (BMS-354 825), that the situation, but most current the existing standard treatment; if it inhibits five tyrosine kinase proteins, models have limited capability for gives better results, it may become including BCR-ABL and SRC (a predicting clinical effects. Models the new gold standard. Phase IV protein that may play a role in that feature biomarker properties, trials are carried out after a drug imatinib resistance). This new agent comparable with those seen in has been licensed. Information is shows very good response in those patient populations, will enhance collected about side-effects, safety who are resistant to imatinib. their utility as predictive models. and the long-term risks and benefits Biomarkers play an important Specific effects on biomarkers in of a drug (http://www.cancerhelp. role in preclinical studies. Critical such models can, in turn, provide org.uk/help/default.asp?page=52). proof-of-concept studies typically proof-of-concept for therapeutic For example, the conventional drug development process will normally Figure 17.3. Roles of biomarkers (grey) and their associated technologies (blue) proceed through all four stages over along the different phases of drug development and post-launch (yellow) many years. A new drug is estimated to cost between US$800 and US$1700 million, and is expected to take anywhere between 7–12 years to be approved and launched. The complexity and duration of clinical trials are determined by the use of a long-term clinical endpoint (e.g. clinical progression, survival) to assess the clinical benefit of a new treatment or drug. Biomarkers have the potential to be used in clinical trials as validated surrogate endpoints to indicate drug efficacy or toxicity, or to make a “go/no-go” decision. Biomarkers can be influential in every phase of drug development, Unit 4 from drug discovery and preclinical evaluations, through each phase of clinical trials and into post- 17 Chapter marketing studies (Figure 17.3). Biomarkers have been used to identify and justify targets for therapy. For example, 95% of CML patients possess a mutation called Philadelphia chromosome, a translocation between chromosome 9 and chromosome 22 that produces a specific tyrosine kinase enzyme, BCR-ABL. Novartis’ Gleevec® Source: (25)

Unit 4 • Chapter 17. Biomarkers in clinical medicine 311 approaches (46). For instance, in be less genetically predisposed UGT1A1 and irinotecan a preclinical study of dasatinib, to respond to the investigational biomarkers Phospho-BCR-ABL/ medication than others. As such, Irinotecan (Camptosar®), a phospho-CrkL were investigated it is typical to enrol large numbers topoisomerase I poison, is approved in K562 human CML xenografts of patients to ensure sufficient for use in combination with grown s.c. in severe combined power to detect with statistical 5-fluorouracil (5-FU) and leucovorin immunodeficient mice. Results certainty any true treatment effect chemotherapy for first-line treatment showed that following a single oral among those who are responsive of metastatic colorectal cancer, and administration of dasatinib at a to the medication. In contrast, also as a single agent in second-line preclinical efficacious dose of 1.25 the application of biomarkers to salvage therapy of 5-FU refractory or 2.5 mg/kg, tumoural phospho- clinical trials enables targeted metastatic colorectal cancer. It is also BCR-ABL/phospho-CrkL were selection of subjects and smaller commonly used to treat esophageal, maximally inhibited at 3 hours and trials by identifying subjects more non-small cell lung cancer, breast recovered to basal levels by 24 likely to respond to a drug based cancer and other solid tumours hours. The time course and extent on their genotype (48). The use in a second- or third-line setting of inhibition correlated with the of biomarkers may lead to more (52). Although it prolongs survival, plasma levels of dasatinib in mice. precise and effective inclusion and it also causes severe diarrhoea Pharmacokinetic/pharmacodynamic exclusion criteria in clinical trials and neutropenia in 20–35% of modelling predicted that the plasma and can be used at multiple points in patients treated. Fatal events during concentration of dasatinib required the drug development process (49). single-agent irinotecan treatment to inhibit 90% of phospho-BCR- Biomarkers will be most valuable have been reported (53,54). UDP- ABL in vivo was 10.9 ng/mL in mice when genotypes for adverse glucuronosyltransferase (UGT1A1) and 14.6 ng/mL in humans, which is response and optimal efficacy for is responsible for the clearance within the range of concentrations a given compound occur at a high by glucuronidation of drugs (e.g. achieved in CML patients who frequency in the patient population irinotecan) and endogenous responded to dasatinib treatment in (50). By applying biomarker-based substances (e.g. bilirubin). As shown the clinic (47). stratification, based on these in Figure 17.4, the primary active and Use of biomarkers can shorten genotypes or protein biomarkers to toxic metabolite of irinotecan, SN- the clinical trial duration. In diseases predict and monitor drug response, 38, is inactivated by UGT1A1 to form with a long natural history, the specific subgroups of subjects SN-38G, which is eliminated via the final result of comparative trials examined in Phase III clinical trials bile. It has been determined that with survival endpoints is often not would be expected to demonstrate variations of the TA repeat length in known for 5–10 years after the study greater response to and/or fewer the UGT1A1 promoter TATA element onset. If these clinical endpoints adverse effects from the drug being may lead to decreased gene could be replaced by validated studied. These trials would likely expression, accumulation of SN-38, surrogate endpoints (biomarkers) decrease drug development time, and irinotecan-related toxicities. that could be measured earlier, costs and potentially speed up the The UGT1A1 gene is located more conveniently or more approval of drugs (51). on chromosome 2q37. The frequently, then new drugs could be polymorphic TA repeat in the validated quicker and administered Examples/case studies 5′-promoter region of UGT1A1 may to patients. In addition, clinical trials consist of 5, 6, 7 or 8 repeats. The could get by with smaller sample The goals of using biomarkers in drug wild-type allele (UGT1A1*1) has six sizes, and costs could be lowered treatment are to minimize toxicity TA repeats, and the variant allele by using stratified patients based on and to maximize the effectiveness (UGT1A1*28) has seven TA repeats. molecular biomarkers. Traditional of therapy. Here are two cases of Patients who are homozygous for drug development relies on the biomarker applications: UGT1A1 for the UGT1A1*28 allele, glucuronidate random assignment of sufficient minimizing toxicity and HER2 for SN-38, less efficiently metabolize numbers of participants with a maximizing drug efficiency. than patients who have one or particular condition to investigational two wild-type alleles; therefore, drug and control groups to enable homozygous patients are exposed detection of statistically significant to higher plasma concentrations of drug responses. Some patients may SN-38 (52,55). In a meta-analysis,

312 Figure 17.4. The irinotecan pathway shows the biotransformation of the chemotherapy of neutropenia; however clinical prodrug, irinotecan, to form the active metabolite SN-38, an inhibitor of DNA results have been variable and such topoisomerase I. SN-38 is primarily metabolized to the inactive SN-38 glucuronide patients have been shown to tolerate by UGT1A1, the isoform catalysing bilirubin glucuronidation. Used with permission from PharmGKB and Stanford University; http://www.pharmgkb.org/do/serve?objId normal starting doses.” (http://www. =PA2001&objCls=Pathway# fda.gov/Drugs/ScienceResearch/ ResearchAreas/Pharmacogenetics/ ucm083378.htm). However, as shown in Figure 17.4, irinotecan interacts with multiple polymorphic drug metabolizing enzymes and transporters (54,56– 62), being inactivated by CYP3A4 to APC and requiring conversion by carboxyesterases to the active metabolite SN38. The latter in turn is inactivated by UGT1A1 glucuronidation as the main degradation pathway. In addition, irinotecan and its metabolites serve as substrates for transporters, including the ABC transporters (ATP-drive extrusion pumps) MDR1, MRP2, and BCRP. Each of these factors displays interindividual variability, with functional polymorphisms potentially contributing to variable irinotecan response. Haplotype analysis has provided additional insight into the regulation of gene transcription (54,56,57,62), but a quantitative assessment of all factors is lacking. As a result, use of TA repeat polymorphisms in predicting in vivo UGT activity and SN38 exposure after irinotecan administration has data presented in nine studies was inclusion of UGT1A1 genotype- been only partially successful. reviewed that included 10 sets of associated risk of toxicity on the The Clinical Pharmacology patients (a total of 821 patients) irinotecan package insert and cites Subcommittee, Advisory Committee and assessed the association of that a clinical test (Invader UGT1A1 for Pharmaceutical Science, Unit 4 irinotecan dose with the risk of Molecular Assay; Third Wave reviewing the product, further noted irinotecan-related haematologic Technologies Inc.) to detect common that “...although there is indication toxicities (grade III–IV) for patients UGT1A1 alleles is available. The to start with a lower dosage, it is 17 Chapter with a UGT1A1*28/*28 genotype FDA-approved label for the test not necessarily an indication that (52). As shown in Table 17.2, the states that “Individuals who are sensitive patients will do well with risk of toxicity was higher among homozygous for the UGT1A*28 allele this dosage” (54). This example patients with a UGT1A1*28/*28 are at increased risk of neutropenia illustrates that pharmacogenetic genotype than among those with following initiation of Camptosar (PGx) testing can identify patients a UGT1A1*1/*1 or UGT1A1*1/*28 treatment. A reduced initial dose who are likely to respond differently genotype at both medium and high should be considered for patients to a particular drug and indicate the doses of irinotecan; however, risk known to be homozygous for the appropriate dosage, but that testing was similar at lower doses (52). UGT1A*28 allele. Heterozygous does not necessarily translate into In 2005, the FDA approved the patients may be at increased risk dosing instructions. Hence, the

Unit 4 • Chapter 17. Biomarkers in clinical medicine 313 Table 17.2. Predictive value of UGT1A1*28 genotype upon irinotegan-induced Grade III-IV haematological toxicity

OXA, oxaliplatin; 5FU, 5-fluorouracil; RAL, raltitrexed; CAP, capecitabine Adapted from the summary table of analyses on 10 clinical trials that assessed the diagnostic value of the homozygous UGT1A1*28 genotype to predict irinotecan-induced grade III-IV hematologic toxicity (52, with permission of Oxford University Press). Other related references are (100–105). value of prospective genotyping for readily accessible, and expression trials were started in 1992. In UGT1A1 in irinotecan therapy must of the receptor is stable in primary advanced breast cancers with HER2 be determined empirically in the tumour tissues and metastatic overexpression, trastuzumab was intended target populations (63). deposits. HER2 became a potential shown to be active as a single agent biomarker with the initial observation in second- and third-line therapy HER2 and trastuzumab that the HER2 gene was amplified in (74,75), and subsequently as first- (Herceptin®) 25% of axillary lymph node-positive line therapy (76). Trastuzumab is breast cancers, and, when present, particularly effective in combination Human epidermal growth factor correlated with poor prognosis (66). with chemotherapy. In 1998, the receptor 2 (HER2), also known as Additional studies confirmed that drug was approved in the United ErbB2 and Neu, is a cell surface HER2 protein overexpression was States by the FDA as Herceptin. glycoprotein with intrinsic TK activity also a prognostic biomarker in breast This drug was able to get a fast that is involved in cell growth and cancer, correlating with decreased track approval status for two development (46,64). In normal relapse-free and overall survival reasons: it demonstrated efficacy quantities, HER2 promotes normal (66–68). Moreover, additional in patients previously resistant to cell growth, but when a genetic clinical data have shown that HER2 more conventional treatments, and mutation causes HER2 to be amplification/overexpression is a a diagnostic test was able to identify overexpressed on the cell surface, predictive biomarker for greater the patients that were expected to certain breast cancer cells are or lesser response to certain benefit from it. The HercepTest is the prompted to multiply uncontrollably chemotherapies or hormonal first example of a pharmacogenomic and invade surrounding tissue therapies in breast cancer (69–73). test that is marketed along with (46,65). The cloned HER2, The role of HER2 as an a drug. There are two tests to associated with a form of metastatic oncogenic protein and clinically determine HER2 status and breast cancer, appeared as a relevant biomarker led directly to the select patients for treatment with potential monoclonal-antibody development of a specific targeted trastuzumab. The first approved was target in 1985. It has many of the therapy: trastuzumab (Herceptin®; an immunohistochemistry (IHC) test, properties required for such a target; Genentech, South San Francisco, the HercepTest, which measures it is overexpressed on the surface of CA), a humanized IgG1 monoclonal the level of expression of the HER2 tumour cells and not on normal cells, antibody with high affinity and protein. The possible outcomes of it has an extracellular domain that is specificity for HER2. The clinical the test are reported as numbers

314 from 0 to 3+, with 0 representing no HER2 positivity could predict the randomized trials. Results of a study overexpression and 3+ representing effect of adjuvant treatment of on trastuzumab use after adjuvant high overexpression. Only 3+ is other drugs. For example, it was chemotherapy in HER2-positive defined as HER2 positive. The reported that HER2 positivity was breast cancer patients found that one most recently approved method, associated with a significant benefit year of this treatment combination fluorescence in situ hybridization from the addition of paclitaxel to had a significant overall survival (FISH), detects the underlying gene the treatment regimen (79). The benefit after a median follow-up of alteration in the patient's tumour interaction between HER2 positivity two years (Figure 17.6) (80). cells. FISH makes the number of and the addition of paclitaxel was A meta-analysis of five HER2/neu gene copies visible. associated with a hazard ratio randomized controlled trials was In healthy cells, there are two for recurrence of 0.59 (P = 0.01). performed comparing adjuvant copies of the HER2/neu gene per Patients with a HER2-positive breast trastuzumab treatment for HER2- chromosome. If FISH detects more cancer benefited from paclitaxel positive early breast cancer. Pooled than two copies of the HER2 gene, it regardless of estrogen-receptor results from the trials showed a means that the cell is abnormal and status, but paclitaxel did not benefit significant reduction of mortality (P is HER2-positive. This abnormality patients with HER2-negative, < 0.00001), recurrence (P < 0.0001), is also referred to as HER2 gene estrogen-receptor-positive cancers. metastases rates (P < 0.0001) and amplification. The results of the The effect of adjuvant second tumours other than breast FISH test can be reported as trastuzumab in the treatment cancer (P = 0.007), as compared to positive or negative. of HER2-positive early breast no-adjuvant-trastuzumab patients Recent comparison of FISH and cancers has been evaluated in (81). IHC shows that FISH appears to randomized controlled trials and However, there are still questions be superior at providing prognostic in a meta-analysis of published about the HER2 biomarker and information with respect to the detection of higher-risk breast Figure 17.5. Algorithm for HER2 testing cancers (77). Unfortunately, it is expensive and requires additional equipment and training beyond what is commonly found in most laboratories. For this reason, it is recommended that only IHC results of 2+ (which represents a little overexpression of HER2) should be retested with FISH to prevent false- negative outcomes (78), as shown in Figure 17.5. The HER2 case is one of the most successful applications of biomarkers in drug development and Unit 4 disease treatment. The advantage of this case is the co-development of drugs and diagnostic tests, which 17 Chapter greatly reduced the number of patients involved in the clinical trials and facilitated a fast-track approval status. It is known that women with HER2+ breast cancer do not respond well to standard therapy, and that patients whose breast cancers lack HER2 overexpression are highly unlikely to respond to trastuzumab alone (46). Moreover, Adapted from http://www.iap-aus.org.au/2001no3.html.

Unit 4 • Chapter 17. Biomarkers in clinical medicine 315 Figure 17.6. Exploratory disease-free survival subgroup analysis for one year of trastuzumab versus observation

Source: (80). Reprinted from The Lancet, Copyright (2007), with permission from Elsevier.

316 trastuzumab treatment. For example, throughput technologies, offer consortium launched two projects: almost half of HER2-positive breast useful tools for biomarker discovery. to address drug-related liver toxicity; cancer patients are non-responsive Genomics studies organisms in and to study a rare but serious drug- to trastuzumab therapy or become terms of their genomes (i.e. their full related skin condition called Stevens- drug resistant during treatment. DNA sequences) and the information Johnson syndrome. The Biomarkers Although other biomarkers have they contain (an indication of what Consortium has launched a web site been investigated, and some drugs can happen). Transcriptomics is to encourage researchers to submit are in clinical trial, no breakthrough used to analyse gene expression biomarker project concepts (http:// drug has been reported yet. Another (what appears to be happening). www.biomarkersconsortium.org/). unsolved issue is toxicity. In the Proteomics is used to investigate In clinical medicine, there are still meta-analysis, they reported more proteins (compounds that make many challenges that must be met grade III or IV cardiac toxicity after things happen). Metabolomics before the full value of biomarkers, trastuzumab (203/4555 = 4.5%) is used to measure metabolites especially molecular biomarkers versus no trastuzumab patients (substances that indicate what has (e.g. cancer biomarkers), can be (86/4562 = 1.8%); therefore, careful happened and is happening). It is realized. cardiac monitoring is warranted (81). widely known that early diagnosis First, identification of highly and effective treatment of common prevalent targets that constitute key Strengths, limitations diseases requires capturing and master promoters of oncogenesis in and lessons learned interpreting information at different specific tumours is still very difficult. levels and using a variety of novel For instance, the oncogenetic Biomarkers have been used in techniques (as shown in Figure 17.3). process in malignant gliomas is disease prevention, diagnosis, Computational technology and driven by several signalling pathways treatment, prognosis and drug bioinformatics play a major role in that are differentially activated or development for many years, the discovery of new biomarkers, the silenced with both parallel and but have only recently shown validation of potential biomarkers, converging complex interactions. To the potential to revolutionise the and the analysis of disease states. date, no new molecule seems to be health paradigm into a new era. For example, Figure 17.7 shows promising enough to justify a large The successful completion of the the detail of a subnetwork of the Phase III trial (82). human genome sequencing project protooncogene MYC. Two types Second, once a potential target laid the foundation for identifying of technologies have made this is identified, it is not easy to discover mechanism-based biomarkers. work possible: the advent of a new agents capable of restoring Although US$1000 per individual new wave of high-throughput normal cell functions through for sequencing is still a ways off, biotechnology, with its sequencers, interaction with the target. A major BioNanomatrix and Complete gene expression arrays, mass hurdle is that tumour cells acquire Genomics Incorporated have spectrometers and fluorescence drug resistance. Certain cancers formed a joint venture to develop a microscopes; and information are effectively treated because the system capable of sequencing the technology for qualitative changes targeted drug is applied. But very entire human genome in eight hours in the way biological knowledge is often patients develop secondary at a cost of less than $100. By its stored, retrieved, processed and mutations that recruit other kinases Unit 4 completion, the proposed technology inferred. that are not affected by the inhibitor to will have the potential to enable Large and well-organized substitute for the pharmacologically improvements in the diagnosis and consortia and networks, as well impaired kinase, and to restore 17 Chapter personalized treatment of a wide as updated regulatory systems, downstream molecular signalling variety of health conditions, as well guarantee the validation of cascades that contribute to tumour as the ability to deliver individually biomarkers and their successful growth (82–84). tailored preventive medicine translation into clinical practice. A Third, there are still many (http://nanotechwire.com/news. good example of this is the FDA methodological issues to resolve. asp?nid=5087&ntid=130&pg=1). consortium that includes members For example, how to define proper Recently developed “omics” of the pharmaceutical industry and criteria for responsiveness, avoid technologies, such as genomics, academia, and aims to observe measurement errors, interpret transcriptomics, proteomics, how genetic biomarkers contribute laboratory results, educate medical metabolomics and other high- to serious adverse events. The staff to accept and use biomarkers in

Unit 4 • Chapter 17. Biomarkers in clinical medicine 317 Figure 17.7. Detail of a subnetwork of the protooncogene MYC. Nodes are colour-coded according to their target status and available validation of direct MYC binding

Source: (107). Reprinted by permission from Macmillan Publishers Ltd: Nature Genetics, copyright (2005). their daily medical practice, and how on those who lack the capacity to Multiple biomarkers will be more to help the public better understand consent, genetic testing on stored frequently applied in clinical tests, genetic tests (85). tissue samples and tissue banks, especially for common diseases. Finally, ethical and social issues and ensuring appropriate monitoring “Multiple” could represent many must be considered. Individual, of genetic tests. These concerns markers from the same profile, or family and societal goals may warrant the attention and action of markers from different profiles, conflict with current health care society as a whole. such as DNA, mRNA, microRNA practices and policies in regards or protein and gene expression. to genetic testing. Current health Future directions In 2007, for example, the FDA care policies do not fully address and challenges approved a gene-based breast these concerns. One major barrier cancer test designed to determine is the potential loss of societal Multiple targets, prevention and the likelihood of early stage breast benefits, such as employment or prediction, personalization and cancer recurrence within 5–10 years insurability, based on one’s genetic cooperation will be the future after treatment. The test called characteristics, which is referred directions of biomarker applications MammaPrintTM (Agendia) is a DNA to as genetic discrimination (85). in clinical medicine. microarray-based diagnostic kit that Other issues include genetic testing measures the level of transcription

318 of 70 genes in breast cancer to have children could be tested in embryos frequently causes failed tumours. The profiles are scored for specific biomarkers to avoid implantation and pregnancy. In a to determine the risk or recurrence haemolytic disease of the newborn recent study, CGH, a genomics and with it the need for adjuvant (HDN) and some recessive diseases approach, was used in assessment therapy (86). There is currently (carrier parents have a 25% chance of embryo aneuploidy and achieved a great deal of research being of passing on the disease to the implantation and pregnancy rates done on multitargeted therapies, baby). Children with a family of 68.9 and 82.2%, respectively which simultaneously target some history of diabetes, heart disease (93). Alternatively, using microarray of the many signalling pathways or cancer may take a genetic test CGH (aCGH) and single nucleotide involved in tumour development to adjust their lifestyle or consider polymorphism microarray have the and proliferation. “Mixing cocktails,” preventive treatment. Therapeutic potential for further improvement in as Charles L. Sawyers recently and prognostic biomarkers should assessment of embryo aneuploidy described it (84), will continue to be applied to all kinds of patients, at a higher resolution, as they can be grow, but should be under the especially cancer patients, to direct used to detect more refined regions appropriate molecular guidance. their treatment plans and predict (less than megabases, or even less Preventive and predictive the treatment outcomes. Within than kilobases of nucleotides) in biomarkers will play a key role in the foreseeable future, when the any chromosome (94,95). Other future health care. New agents, US$100 genome sequencer is omics have also been applied such as antiangiogenesis/vascular- developed, everybody would be to assessing embryo viability, targeting drugs, have moved from able to have their whole-genome such as metabolomics (96,97), cancer therapy to cancer prevention. information on their ID card. transcriptomics (98) and proteomics Molecular and epidemiologic In the first decade of the 21st (99). These omics technologies studies of cancer risk and drug century, the fast-growing application present unique advantages as well sensitivity and resistance began of omics technologies in translational as their own intrinsic limitations. ushering in the era of personalized research and clinical medicine have However, a combined strategy of prevention (84,87). Development been witnessed. It has accelerated omics may enhance the thorough of new treatments has increased biomarker development, improved screening of gametes and embryos the need for markers that predict the accuracy for diagnosis/treatment, for their viability and reproductive outcome and those that direct which and advanced personalized potential. The applications of omics treatment options are most likely to medicine. One example is the technologies in other medical be effective for a particular patient application of omics in reproductive fields are in different stages of with a particular tumour (88). medicine, in particular in vitro development and ever expanding. Personalized medicine is the fertilization (IVF) treatment, an It is envisioned that the biomarkers use of detailed information about assisted reproduction. A key step derived from those omics will realize an individual’s inherited and/ in assisted reproduction is the their full potential before long in all or acquired characteristics and assessment of oocyte and embryo fields of clinical medicine. their phenotypic data to select a viability to determine the embryo(s) In summary, biomarkers preventative measure or medication most likely to result in a pregnancy. have been widely used in that is particularly suited to that Although conventional systems such clinical prevention, diagnostics, Unit 4 person at the time of administration. as morphological charaterization therapeutics, prognostics, clinical This revolution in clinical care is and cleavage rating have been trials and drug development. With predicated on the development and successful in improving pregnancy mapping of the human genome 17 Chapter refinement of biomarkers, enabling rates, their precision is far from ideal complete, rapid development of new disease prevention, and diagnosis (90,91). It was reported that two out technologies and the collaboration and treatment of patients and of three IVF cycles fail to result in of different disciplines, biomarkers populations (89). Biomarkers will be a pregnancy, and more than eight promise personalized medicine, used before birth and throughout out of 10 embryos fail to implant though many challenges remain to life. For example, a couple planning (92). The presence of aneuploidy be overcome.

Unit 4 • Chapter 17. Biomarkers in clinical medicine 319 References

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