Current Topics in Genetics Vol. 5, 2012

Companion diagnostics and : A review of molecular diagnostic applications

1 2 1, Mathew W. Moore , Deepti Babu , and Philip D. Cotter * 1 2 ResearchDx, Irvine, California, USA. Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada

ABSTRACT well established [1, 2]. The advantages of approaching medicine in this way are theoretically Personalized medicine is the customization of treatment based on a patient’s hereditary or clear; personalized medicine has the potential to somatic genetics and holds the promise of more efficiently, effectively, and safely direct revolutionizing healthcare. Companion diagnostics, health care than traditional non-targeted approaches. many of which are molecular genetics assays, are While the rate of progress has clearly increased, there are still significant technical and regulatory critical tools in the implementation of personalized medicine. Information derived from these tests hurdles to overcome. Several guidance documents provides for customizing specific therapies based from regulatory organizations worldwide have on the genetics of the disease. While the benefits attempted to address these challenges, and no are clear, the path to a successful companion doubt more will be presented in the near future [3]. diagnostic has required a forging of new alliances between drug and diagnostic developers, clinical Companion diagnostics are increasingly relied laboratories, physicians, pathologists, and healthcare upon to ensure the effective, safe development providers. Molecular genetic companion diagnostic and use of a personalized therapeutic. Multiple assays are becoming more relevant and important liaisons and partnerships between key stakeholders in an environment of increased regulatory guidance are needed in this complex, dynamic process. in their development and application. Here, we Many successful companion diagnostics are review key molecular genetics companion diagnostic genetic tests - particularly molecular diagnostics - tests and their applications in personalized medicine. and this speaks to their high impact and relevance in the field of personalized medicine. KEYWORDS: personalized medicine, genetic, molecular, companion diagnostics Personalized medicine and the “new” genomics

“Personalized medicine” is a phrase first coined in INTRODUCTION the 1990s, although the concept pre-dated this The concept of personalized medicine, whereby [4, 5]. Achieved successfully, personalized medicine disease diagnosis, treatment and prevention are harnesses power from innate biological information customized to one’s genetic composition, is now to direct appropriate therapies for appropriate patients - a goal that maximizes key components of effectiveness, efficiency, and safety. *Address correspondence to: Dr. Philip D. Cotter, ResearchDx, 13766 Alton Parkway, Suite 147, Independent of the Human Project, Irvine, CA 92618, USA. personalized medicine efforts initially began with [email protected] a consortium of the world’s largest pharmaceutical

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companies and scientists, created to identify Early strategic partnerships are necessary to create natural genetic differences between people [4]. personalized therapeutics. Few pharmaceutical The goal was to correlate informative genetic companies have depth of experience in the with disease symptoms, or serious diagnostics arena. Partnerships can address side effects to certain medications. Drug experimental design, discovery, assay developers then hoped to develop more effective, validation, marketing and commercialization [3]. safer drugs to target these patient populations. However, some of these partnerships are not part

More recently, the interest in personalized of current pharmaceutical company outsourcing medicine has increased substantially; based on practices. In addition, early alliances are difficult PubMed searches on the term ‘personalized because the value of the drug and its diagnostic medicine’, a 2011 publication found that the are difficult to predict [3]. These and other factors bring about several business challenges. number of scientific publications on the subject has shown an exponential growth in the period Commercially, there are several challenges for a from 1999 to 2010 [6]. Kongkaew et al. [7] companion diagnostic. The total market for the estimated that more than 5% of hospital therapeutic needs to be large enough to not only admissions are associated with adverse reactions justify the development cost of the therapeutic to prescribed drugs. Many of these are due to itself, but also now the cost of development for individual genetic differences that render one the associated companion diagnostic. In addition, hypersensitive to the drug, or unable to one has to market the value of both the therapeutic metabolize it properly [8]. and the diagnostic. A companion diagnostic has the potential to reduce the market size for a Challenges in the drug development process therapeutic by limiting the patient population. Responses from the larger pharmaceutical and Similarly, in an extreme case the companion biotechnology companies to create personalized diagnostic may leave the physician without a therapeutics have been lower than expected, based viable therapeutic treatment. These issues can on interest level. Success rates in bringing these make pharmaceutical companies less commercially drugs to market have also been low. A number of motivated to pursue a personalized therapeutic. scientific, strategic, commercial, and regulatory Regulatory factors can also bring challenges, factors have been attributed to this [9]. as there are inefficiencies in the current drug Jorgensen et al. [6] argued that the initial “one development process. The co-submission of a drug for one disease” model does not fit the therapeutic and diagnostic complicates the clinical reality of heterogeneous disease regulatory submission process and can lead to mechanisms at the molecular level. As a result, increased costs and delays. some diseases have not been as amenable to Historically, there were few regulatory guidance personalized medicine, as was initially postulated. documents to manage drug and companion Scientifically, it has also been more difficult to diagnostic co-development. More recently, identify and validate biomarkers in as timely a regulatory agencies have responded with guidance fashion than the industry initially expected. While documents, attempting to inform best practices, the drug development and regulatory process is and to provide clarity and consistency in assay well known and understood, the development of a development and marketing approval [3]. successful requires an understanding of several success factors including biomarker availability, robust technical assay validation, the Regulatory responses about companion diagnostics importance of demonstrating clinical utility, and the ability to bring an investment-positive Regulatory agencies are quickly recognizing that commercial value proposition to the table [3]. companion diagnostics can be the key to a safe, This is extremely difficult to find without successful personalized therapeutic. In draft partnering with several organizations, introducing guidance from July 2011, the FDA indicated that, several logistical challenges to the process. “in most circumstances, if use of an in vitro

Genetic tests as companion diagnostics 25

companion diagnostic device (IVD companion the colon, lung, breast and other sites [1]. These diagnostic device) is essential for the safe and companion diagnostics typically identify somatic effective use of a therapeutic product, (it and its) mutations identified in tumor cells, which help therapeutic product should be approved or cleared direct use of an appropriate therapeutic (Table 1). contemporaneously by FDA for the use indicated in the therapeutic product labeling” [10]. The Recent successful genetic companion diagnostic guidance also stated that, “the results of the IVD tests in companion diagnostic device will be essential for Crizotinib and non-small cell lung the safe and effective use of the therapeutic product, and its use will be stipulated in the Recently, rearrangements of the anaplastic labeling of the therapeutic product.” Because the lymphoma kinase (ALK) gene were reported in non-small cell lung cancer (NSCLC) [16, 17]. IVD companion diagnostic was identified as essential for this purpose, it was noted that, “with Within three years, studies of ALK inhibition some exceptions FDA does not believe it may yielding dramatic response rates in patients with approve a novel therapeutic product or new advanced NSCLC containing ALK rearrangements therapeutic product indication for use with an IVD were reported [16, 18, 19]. In pretreated patients companion diagnostic if the IVD companion that generally have a 10% response rate to conventional chemotherapy, treatment with the diagnostic is not approved or cleared for that ® indication” [10]. Guidelines for the development oral ALK inhibitor crizotinib (Xalkori ) yielded of IVDs also exist in the European Union (EU) an overall response rate of 55% and an estimated [11]. six-month, progression-free survival rate of 72% [16]. Regulatory guidances put an increased focus and relevance on the development of companion Significantly, the mechanism of resistance was associated with ALK kinase domain mutations, diagnostics, many of which are genetic tests. Gene-based and molecular diagnostics testing is substantiating that ALK was indeed the genetic growing at a 30-50% rate, and it has been target of the personalized therapy [16]. This also estimated that as many as 1,500 genes and 5,000 reinforced that appropriate clinical application of may be candidates for new molecular test ALK-targeted therapy was absolutely dependent targets [12, 13]. It has also been recommended upon a companion diagnostic to identify patients most likely to respond. The FDA has since that companion diagnostics be used at an early stage in the drug development process [9]. From approved the drug, and requires use of its a financial perspective, molecular diagnostics companion diagnostic; this is indicated in product labeling. within the USA alone was valued at approximately $2.7 billion in 2006, and was expected to reach Vemurafenib and metastatic malignant $5 billion by 2010 (AGR 15%) [14]. Oncology melanoma molecular diagnostics was the fastest growing The B-Raf proto- serine/threonine- sector at that time and was predicted to increase kinase (BRAF) gene is critical in the by 30% each year, tripling from its 2005 level of development of melanoma [20, 21]. Melanoma $315 million to more than $1.35 billion by 2010 tumor cells with BRAF mutations contain [2, 14]. distinctive characteristics, such as unique morphological variants, an age at diagnosis often Personalized medicine in the oncology sector before 55 years, and others [21]. A multi-centric There has been significant progress for study reported that the treatment of metastatic companion diagnostics and personalized medicine melanomas carrying the V600E mutation in in the oncology sector. For example, the use of BRAF with a selective small molecule inhibitor pre-symptomatic and “targeted PLX4032 (vemurafenib) resulted in complete or therapies” tailored to genetic profiles of tumors is partial regression of disease in most patients part of a recommended evaluation for of [20, 22]. From clinical trial studies, patients with

6 MathewW.Moore 26 Table 1. Oncology companion diagnostics required by the FDA.

Genetic biomarker Required companion Drug FDA approved Drug manufacturer diagnostic t(15;17) chromosome translocation or Vesanoid® (tretinoin), PML/RARα gene expression in acute PML/RARα Trisenox® (arsenic 1995, 2000 Roche, Cell Therapeutics, Inc. promyelocytic leukemia trioxide) Overexpression of HER-2 in metastatic Herceptin (trastuzumab), HercepTest™ (1998) 1998, 2007 Genentech, GlaxoSmithKline breast cancer tumor cells Tykerb (lapatinib) Philadelphia chromosome positive [BCR-ABL+] chronic myeloid leukemia BCR-ABL Gleevec® (imatinib) 2001 Novartis in chronic phase Platelet-derived growth factor receptor (PDGFR) gene rearrangements in 2001, (2006 this PDGFR Gleevec® (imatinib) Novartis myelodysplastic/myeloproliferative indication) diseases Hodgkin’s lymphoma cells expressing CD20 Bexxar® (tositumomab) 2003 Corixa CD20 antigen High expression of EGFR (more likely DakoCytomation EGFR Erbitux® (cetuximab), 2004, 2006 Eli Lilly, Amgen to respond) pharmDx™ test kit Vectibix® (panitumumab) Chromosome 5q deletion associated with transfusion-dependent anemia due to low- or intermediate-1-risk MDS, 5q deletion Revlimid® (lenalidomide) 2005 Celgene Corporation with/without additional cytogenetic abnormalities Philadelphia chromosome-positive acute lymphoblastic leukemia with BCR-ABL Sprycel® (dasatinib) 2006 Bristol-Myers Squibb resistance or intolerance to prior therapy Expression of CD25 component of IL-2 Ontak® (denileukin receptor in persistent or recurrent CD25 2008 Eisai Medical Research diftitox) cutaneous T-cell lymphoma ELM4-ALK translocation-positive ELM4-ALK advanced or metastatic non-small cell Xalkori® (crizotinib) 2011 Pfizer translocation lung cancer BRAF V600E mutation in unresectable BRAF V600E mutation Zelboraf™ (vemurafenib) 2011 Genentech (Roche) or metastatic melanoma et al. Sources: [6, 9, 15]. Genetic tests as companion diagnostics 27

BRAF V600E mutation-positive melanoma drug metabolism. Metabolism in the liver by receiving vemurafenib (Zelboraf™) showed cytochrome P450s represents the most common improved rates of overall and progression-free route of drug breakdown. Fast- and slow- survival, as compared to those receiving metabolizing variants due to mutations in these conventional therapy. This highlighted the enzymes can lead to under- and over-dosing of importance of a molecular disease model focusing drugs [8, 28]. The FDA approved Roche’s on specific biomarkers, identified by companion AmpliChip™ microarray-based assay to identify diagnostics, as bona fide targets that could benefit 29 variants in the two most common drug- melanoma patients [21]. The FDA subsequently metabolizing P450s: CYP2D6 and CYP2C19. approved the drug and requires use of the Known to mediate the metabolism of almost 25% companion diagnostic prior to its administration; of drugs, adverse events with nearly 30 drugs are this is indicated in product labeling. known to be related to drug accumulation in patients carrying variants in these two enzymes Key companion diagnostics outside the [8, 29]. oncology sector Recently, findings were published on the prediction Genetic biomarkers identify risk for of dose selection for warfarin after correlation life-threatening drug side effects with genomic data and a pharmacogenetic Companion diagnostics for hereditary mutations algorithm [8, 30]. Under- or over-dosing with are also becoming more widespread (Table 2). warfarin is the worldwide leading cause of Heterozygosity for the hospitalization related to adverse events. Variants HLA-B*1502 allele, found almost exclusively in in CYP2D9 and VKORC1 are known to influence individuals from some parts of Asia, placed one the biologic breakdown of warfarin. Study results at increased risk of life-threatening reactions have shown that the prediction of dose selection to carbamazepine, a commonly prescribed anti- with a pharmacogenetic algorithm correlated well convulsant in those geographical regions [25]. with empirically determined maintenance doses. Once the risk for these serious reactions in those In fact, this outperformed clinical prediction and with the HLA-B*1502 allele became known, the standard dose estimates. This was particularly true FDA issued an alert, indicating that if an in the outlier population; patients with common individual tests positive for the allele … variants of the metabolizing enzymes fell within “carbamazepine should not be started unless the the range of standard dosing [8, 30]. The FDA has expected benefit clearly outweighs the increased been updating drug labels to include such genetic risk of serious skin reactions.” (http://www. information where compelling data exist [8]. fda.gov.login.ezproxy.library.ualberta.ca/Drugs/D rugSafety/PostmarketDrugSafetyInformationforPa New alliances are needed to ensure successful tientsandProviders/ucm124718.html). The FDA companion diagnostics then recommended an HLA-B*1502 companion The pathway to a successful companion diagnostic to be performed prior to prescribing diagnostic is complex, and is best achieved carbamazepine to those from at-risk populations. through a closely coordinated interaction between A 2011 letter to the New England Journal of diagnostic manufacturers, drug companies and Medicine augmented this warning by stating, “Given regulatory agencies [11]. More specifically, this the availability of other elective therapeutic requires alliances between drug and diagnostic choices, it may be prudent to advise HLA-B*1502 developers, clinical laboratories, physicians, carriers to avoid not only carbamazepine but also pathologists, healthcare providers, and others other structurally related anticonvulsants, such as (Figure 1). phenytoin, oxcarbazepine, and possibly lamotrigine.” [26, 27]. Although companion diagnostics have been developed and approved in some therapeutic Drug metabolism variances due to hereditary areas, the regulatory process itself is still evolving. mutations Each companion diagnostic case is unique and Other successful companion diagnostics are requires early interactions and planning to ensure associated with hereditary mutations that affect an efficient path to market [11].

MathewW.Moore 28 Table 2. Companion diagnostics outside the oncology sector required or recommended by the FDA. Drug Manufacturer Indication(s) Companion diagnostic Trofile™ (Monogram Treatment of only CCR5-tropic human immunodeficiency Biosciences) tropism assay for Selzentry® (maraviroc) Pfizer virus (HIV) in adults, in combination with other antiretroviral CCR5 receptor - required by the agents. FDA Treatment of bipolar disorder, depression, seizures, autism, chronic pain with neuropathy, migraine headaches. Metabolic and/or molecular Hyperammonemic encephalopathy (sometimes fatal) has been Depakote® (valproic acid) Abbott Laboratories testing for urea cycle disorders - reported following initiation of valproic acid therapy in recommended by the FDA patients with urea cycle disorders, particularly ornithine transcarbamylase deficiency (OTC). Anticoagulant used to reduce the risk for or treat thrombosis, CYP2C9 and VKORC1 or as secondary prophylaxis to reduce the risk for embolism. Coumadin® (warfarin) Various genotyping - recommended Those with certain mutations in CYP2C9 and VKORC1 P450 by the FDA enzymes metabolize warfarin differently. LDLR, APOB, PCSK9 mutation Treatment of hypercholesterolemia (familial and non- analysis (for autosomal dominant Lipitor® (atorvastatin) Pfizer familial). Those with homozygous and heterozygous familial forms) - recommended by the hypercholesterolemia need to receive specific doses. FDA Treatment of seizures. Severe dermatologic reactions are HLA-B*1502 genotyping - Tegretol® (carbamazepine) Various associated with HLA-B*1502 allele, found in some Asian recommended by the FDA patients. Treatment of chronic hepatitis C with compensated Pegasys® (peginterferon liver disease. A single nucleotide polymorphism (SNP) near IL28B genotyping - Genentech alfa-2a) the gene encoding interferon-lambda-3 (IL28B) is associated recommended by the FDA with variable sustained virological response rates. Antiretroviral treatment of HIV. Patients who carry the HLA-B*5701 genotyping - Ziagen® (abacavir) GlaxoSmithKline HLA-B*5701 allele are at high risk for experiencing a recommended by the FDA hypersensitivity reaction to abacavir. Antiparasitic treatment of malaria; can also be used to treat Metabolic and/or molecular lupus. Increased risk of hemolysis when administered to Aralen® (chloroquine) Various testing for G6PD deficiency - patients with glucose-6 phosphate dehydrogenase (G6PD) recommended by the FDA deficiency. Sources: [23, 24]. et al. Genetic tests as companion diagnostics 29

Figure 1. Necessary alliances to develop a successful companion diagnostic.

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