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P T E R

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Analytical Methods and BiomarkerValidation

Haleem J. Issaq, Timothy D. VeenstraLaboratory of Proteomics and Analytical Technologies, Advanced Technology Program, Frederick

National Laboratory, Frederick, MD, USA

Ph

O U T L I N E

Introduction 44

7

Discussion 448

roteomttp://

Analytical Method Validation

448

Experimental Design and Execution 448

Biomarker Identification and Confirmation 449

Biomarker Validation 449

Phase 1: Preclinical exploratory studies to

identify potentially useful markers

449

ic and Metabolomic Approaches to Biomarker Discoverydx.doi.org/10.1016/B978-0-12-394446-7.00028-5 447

Phase 2: Clinical assay development forclinical disease

449

Phase 3: Retrospective longitudinal repositorystudies

449

Phase 4: Prospective screening studies

450 Phase 5: Cancer control studies 450

Conclusions 450

References 450

INTRODUCTION

Graduating a discovered potential biomarkerfrom the laboratory to the clinic requires carefulconfirmationandvalidation.Validationofa candi-date protein ormetabolite biomarker involvesfivemajor steps, as discussed later in this chapter.However, clinical evaluation of a biomarker doesnot mean that validation applies only to the

candidate biomarker. Validation also applies tothe experimental steps leading to the biomarker’sdiscovery, including study design, experimentalsetup, data acquisition, data processing, and inter-pretation of the results. Analytical validationinvolves instrument qualification and methodvalidation. Instrument qualification verifies thatthe selected instrument is suitable for its intendeduse, has been qualified by the manufacturer, and

Copyright � 2013 Elsevier Inc. All rights reserved.

28. ANALYTICAL METHODS AND BIOMARKER VALIDATION448

meets the acceptance criteria for all parameters.Method validation verifies that the optimumanalytical parameters that provide high reproduc-ibility and accuracy have been utilized. Duringroutine analysis, the instrument and methodshould be periodically verified to demonstratethat they meet the acceptance criteria. Analyticalinstrument qualification, performance qualifica-tion, and operational qualification were discussedin a recent review by Clanciulli and Watzig1 forcapillary electrophoresis and the points theydescribed are applicable to other types of analyt-ical instrumentation. It is important that the exper-iment be run at least in duplicates to insure thereproducibility of the results, because an error inthe identification of a protein or metabolitebiomarker means a loss of time, effort, andresources spent on the validation process. Thenext phase in biomarker discovery is the valida-tion of the candidate biomarker, which involvesfive phases, as discussed in detail by Pepe et al.,2

Khleif et al.,3 andChau et al.4 After the two phasesof discovery and validation have been reproduc-ibly and successfully completed, a biomarker canbe moved from the laboratory to the clinic.

DISCUSSION

Analytical Method Validation

The search for a biomarker is not a simplematter and involves multiple steps and differentpersonnel with different qualifications: a physi-cian to confirm the presence or absence of thedisease, a surgeon to remove the tumor, a pathol-ogist to examine and certify the tumor type,a technician to prepare the sample, a chemist orbiochemist to design and perform the experi-ment, a statistician to interpret the data, and anepidemiologist to validate the candidate proteinor metabolite biomarker in a larger population.The selected method should be reliable, provideaccurate and reproducible results, and bespecific for the detection and quantitation ofthe protein or metabolite biomarker.

The design of a study involves many stepsthat need to be considered before a search fora biomarker can be initiated. These include:

• Selection of a disease.• If the selected disease is a cancer, the type

and stage should be clearly identified andconfirmed by both a certified physician and apathologist.

• Number of patients and controls who haveundergone a clinical examination.

• Number of specimens needed to carrya meaningful study.

• The characteristics of the individuals whoprovided samples for the study (e.g., age, sex,ethnicity, etc.).

• Type of samples, such as tissue or biologicalfluid and their storage history.

• If using biofluids, the type of biofluid (e.g.,blood, urine, saliva, etc.).

• If the biofluid is serum or plasma, type ofpreservative used.

• Whether the search is for a protein ormetabolite biomarker.

• Method of sample collection, handling,storage, and shipment.

• Method of analysis (e.g., MS, NMR, etc.).

EXPERIMENTAL DESIGN ANDEXECUTION

After the disease to study has been selectedand the samples are ready for analysis, it istime to decide on all aspects of the experiment.It is a good practice to have a standard operatingprocedure (SOP) in place that is written specifi-cally for the study under consideration. TheSOP should specify the following:

• Method of analysis (e.g., NMR, MS,HPLC, GC, CE, SDS-PAGE, 2D-DIGE,etc.).

• Matrix specific sample preparationprocedure.

• Extraction procedures, if necessary.

BIOMARKER VALIDATION 449

• Metabolite derivatization procedure for GCanalysis.

• Protein digestion conditions.• Instrument qualification; ensure that all

instrumentation is operational andperformance qualified by a certifiedtechnician.

• Operator training and qualification.• Analytical procedures should be validated,

inter- and intraday variability, sample tosample reproducibility and relative standarddeviation should be determined and theiracceptable levels specified.

• Reproducibility: the results should bereproducible in other labs, by differentpersonnel using different (but equivalent)instrumentation.

• Blind testing and interpretation of results.• Identities and purity of internal standards

should be confirmed.• Determine whether different techniques

measure the same compounds quantitatively.• Statistical data analysis method should be

specified.

BIOMARKER IDENTIFICATIONAND CONFIRMATION

Analytical and statistical data determined thatcertain proteins or metabolites are found to bedifferentially expressed in the biological fluid ortissue of the patient subjects compared to healthycontrols. At this point, a rigorous investigation ofthe results should be undertaken to confirm theidentity of the potential biomarker. The analyticalmethods of choice for the identification of proteinsor metabolites biomarkers include LC/MS, gelelectrophoresis/MS, and NMR. In the case ofLC/MS, the compounds are identified by theirmass/charge (m/z) ratio and retention time.Proteins are identified by MS/MS, owing to theinvaluable sequence information it provides.Because of isobaric compounds, metabolites maynot be as easy to identify as peptides and proteins.

The identities of metabolites are generally basedon comparison of the experimental data to thatcontained within databases, followed by compar-ison to a pure compound of the same identity. Thecomparison should involve more than one proce-dure or parameterdfor example, m/z ratio, reten-tion time, sequence, NMR resonance.

BIOMARKER VALIDATION

Validation of a biomarker is a lengthy and costlyprocess that involves a multiphase approach.Pepe et al. 2 suggested that the followingfive phasesbe carried out consecutively.

Phase 1: Preclinical exploratory studiesto identify potentially useful markers

This step involves the experimental identifica-tion of a protein or metabolite as a discriminatingfactor between diseased samples and controls.Most published proteomic and metabolomicstudies stop at this phase creating a glut ofpotential biomarkers that languish at this phase.

Phase 2: Clinical assay development forclinical disease

The aim of this phase is to ensure that thecandidate biomarker (protein or metabolite)discriminates between diseased and controlsamples that were obtained in a noninvasivemanner. This phase also involves optimizationof the analytical method. It is important to deter-mine the effect of sex, age, and ethnicity ofcontrols on the biomarker discovery resultswhileassessing the ability to differentiate betweencharacteristics such as cancer stage and grade.

Phase 3: Retrospective longitudinalrepository studies

The primary aims of this phase is to evaluate,as a function of time before clinical diagnosis, thecapacity of the biomarker to detect preclinical

28. ANALYTICAL METHODS AND BIOMARKER VALIDATION450

disease and to define criteria for a positivescreening test in preparation for the next phase.2

Phase 4: Prospective screening studies

This phase, unlike the previous three phases, isapplied to samples taken from individuals andnot on stored specimens. The primary aim is todetermine the operating characteristics of thebiomarker-based screening test in a relevant pop-ulation by determining the detection rate andfalse referral rate.2 The sensitivity and specificityof the biomarker is determined at this phase.

Phase 5: Cancer control studies

The aim of this phase is to determine the valueand effectiveness of the biomarker as a screeningclinical test on the number of cancer cases ina population. Does screening help reduce thenumber of cancer cases? The most effective anduseful biomarker is one that can detect the diseaseat an early stage, prior to clinical symptoms.2

CONCLUSIONS

The search for a protein or metabolitebiomarker in biological fluids and tissues is chal-lenging and involves people with different typesof expertise and analytical techniques. The resultsmust be confirmed and validated and should befree from overfitting of the data (which canhappen when a large number of variables aremeasured on a small number of samples) andshould be free of bias. The method used to vali-date a biomarkermust also be accurate, sensitive,and reproducible.5 The instrumentation needs tomeet specified criteria and be qualified for itsintended purpose. Using urothelial cancer as anexample, Droller6 exquisitely summarizes the

definition of validation that applies to cancerand other diseases:

“Markers for urothelial cancer must beexpressed exclusively as a consequence of thepresence of cancer cells. The testmust be sensitivein detecting disease and must be validated innon-selected populations. It must be sensitive inexcluding non-disease, and non-cancer condi-tions must have little influence on its accuracy.There must be little or no inter-assay or intra-assay variability. Interpretation of an assay fora tumor marker should be all or none, or basedon a threshold level. The assays must have suffi-cient sensitivity and specificity in populationsand in the individual to have practical clinicalapplicability.”Finally,“abiomarker is consideredvalidatedwhen the factors identified in a trainingset of patients give a statistically significant asso-ciation in an independent validation cohort con-ducted in an independent laboratory.”7

References1. Clanciulli C, Watzig H. Analytical instrument qualifica-

tion in capillary. Electrophoresis 2012;33:1499e508.2. Pepe MS, Etzioni R, Feng Z, et al. Phases of biomarker

development for early detection of cancer. J Nat CancerInst 2001;93:1054e61.

3. Khleif SN, Doroshow JH, Hait WN. AACR-FDA cancerbiomarkers collaborative consensus report: advancingthe use of biomarkers in cancer drug development. ClinCancer Res 2010;16:3299e318.

4. Chau CH, Rixe O, Mcleod H, et al. Validation of analyticmethods for biomarkers used in drug development. ClinCancer Res 2008;14:5967e76.

5. Brenner DE, Normolle DP. Biomarkers for cancer risk,early detection, and prognosis: the validation conun-drum. Cancer Epidemiol Biomarkers Prev 2007;16:1918e20.

6. Droller MJ. Current concepts of tumor markers inbladder cancer. Urol Clin North Am 2002;29:229e34.

7. Roos PH, Golka K, Hengstler JG. Predictive biomarkersand signatures in urinary bladder cancer. Cur Opn MolTherapeutics 2008;10:243e50.