Polymerase Chain Reaction

C H A P T E R

6 Polymerase Chain Reaction Karen Yu, Sylwia Karwowska, Abha Sharma, Oliver Liesenfeld and Sidney A. Scudder Medical and Scientific Affairs, Roche Molecular Diagnostics, Pleasanton, CA, United States O U T L I N E 6.1 Polymerase Chain Reaction Technology 111 6.1.1 The Invention of Polymerase Chain Reaction—The Principle, Its Invention and 35 Years of Development 112 6.1.2 Different Polymerase Chain Reaction Techniques—A Multitude of Opportunities for Companion Diagnostics 113 6.1.3 Polymerase Chain Reaction Versus Other Diagnostic Technologies 115 6.2 Companion Diagnostics for Oncology Applications

6.3 Development Process 6.3.1 General Considerations 6.3.2 Analytical Verification and Validation 6.3.3 Clinical Validation 6.3.4 Considerations for the Statistical Analysis of Companion Diagnostics Clinical Studies

120 120

6.4 Clinical Study Conduct

128

6.5 Summary/Conclusion

132

References

133

121 124

125

116

6.1 POLYMERASE CHAIN REACTION TECHNOLOGY Since its inception in 1983, polymerase chain reaction (PCR) has been used across many fields of biological science to allow the study of an individual’s DNA. Whether determining the pedigree of animals or humans, to verifying paternity of a child or exonerating someone wrongly accused of a crime, PCR has permeated our everyday lives. Using PCR, scientists have been able to analyze DNA for a broad range of medical purposes; among others,

Companion and Complementary Diagnostics DOI: https://doi.org/10.1016/B978-0-12-813539-6.00006-7

111

© 2019 Elsevier Inc. All rights reserved.

112

6. POLYMERASE CHAIN REACTION

matching of donor organs with recipients in transplant programs, detection and monitoring of pathogens in infected patients and blood and tissue donors, detection of fetal pathologies in maternal blood, and the detection of blood disorders and solid organ malignancies. In this chapter, we will highlight the history of PCR as it relates to diagnostics with a focus on companion and complementary diagnostics. The US Food and Drug Administration (FDA) approvals for companion diagnostic (CDx) tests and complementary diagnostics are discussed. We will also provide an in-depth overview of the design of analytical and clinical studies required for the evidence gathering and regulatory approval of CDx and complementary diagnostics with a particular focus on requirements for the successful conduct of clinical studies at the core of registration with regulatory bodies. This chapter will provide the reader with an introduction to the topic; key references are presented to allow the interested reader a deeper dive into the topic.

6.1.1 The Invention of Polymerase Chain Reaction—The Principle, Its Invention and 35 Years of Development If there is a single scientific discovery that gave birth to molecular diagnostics, this would be the invention of the PCR in 1985. PCR uses the principle of DNA replication observed in nature and requires a DNA sample, DNA polymerase, nucleotides, target-specific primers, and other reagents in a reaction tube to copy the DNA code. The principle, as first reported in 1987, consists of three steps in a single PCR cycle, which doubles the amount of target DNA. After multiple cycles with exponential multiplication of target DNA, millions of copies of the target DNA in the original sample can be generated (Fig. 6.1).

Original DNA to be replicated

5⬘ 5⬘

3⬘

5⬘

3⬘

3⬘ 3⬘

5⬘

1

3⬘

5⬘

3⬘ 2

3

5⬘ 5⬘ 3⬘ DNA primer

1

1

2

2

3

3

3⬘

5⬘ 3⬘

5⬘

3⬘

5⬘

Nucleotide

1

Denaturation at 94°C–96°C

2

Annealing at ~68°C

3

Elongation at c. 72°C

FIGURE 6.1 Test principle of polymerase chain reaction. 20
40 repeat cycles consisting of three steps (denaturation, annealing, and elongation) are performed at varying temperatures. Specific temperatures and the length of each cycle are adjusted depending on the targets to be amplified by the reaction (https://en.wikipedia.org/ wiki/Polymerase_chain_reaction).

COMPANION AND COMPLEMENTARY DIAGNOSTICS

6.1 POLYMERASE CHAIN REACTION TECHNOLOGY

113

First, following sample preparation to isolate DNA (often using magnetic glass beads which bind negatively charged nucleic acids), the PCR reaction encompasses three sequential steps. First, denaturation is required to separate the double-stranded target DNA, typically achieved by heating the sample to more than 90 C to break the noncovalent bonds between the DNA-forming nucleotides. Second, annealing of the target DNA sequences needs to occur. This is achieved by binding of PCR primers which are complementary to the specific DNA sequences of interest in the sample. Primers are specifically designed oligonucleotides that selectively bind to sequences on either side of the target DNA region; one primer each binds to the start of the target sequence in the newly separated single DNA strands. The binding of primers typically occurs at temperatures between 40 C and 60 C. Finally, extension of the target sequences with the bound primers is performed to generate a copy of the target sequence at temperatures around 72 C. This is achieved by a DNA polymerase which uses added nucleotides to create a new strand of DNA complementary to each of the single template strands. Having produced two copies of the DNA through the first cycle of PCR, the sequence of steps is restarted, and the newly duplicated DNA can be copied over multiple cycles. Applying the complementarity of DNA with exquisite sequence specificity due to primers and the process of DNA replication, a DNA “photocopier” in a test tube was born. These experimental concepts and the availability of thermostable DNA polymerases allowed exponential amplification of DNA target sequences and detection with labeled oligonucleotide probes [1]. In 1992 the AMPLICOR Chlamydia trachomatis test was introduced outside of the United States, followed a year later with 510(k) clearance by the FDA, making it the first FDA-cleared diagnostic in vitro diagnostic (IVD) test based on PCR technology. In 1993 Kary Mullis won the Nobel Prize in Chemistry for his discovery of PCR. Advances in technology led to the development of real-time (RT) PCR [2], during which the DNA produced in the PCR amplification reaction is monitored in RT using fluorescent probes to detect the presence of the target sequence. As the amplified target DNA accumulates with every PCR cycle, the amplified DNA segments “light up,” allowing a quantitative detection of the target. Because PCR and detection could now be completed simultaneously, not sequentially, the time to result was decreased and human interaction with the samples was decreased to a minimum thereby lowering the risk of contamination and making such tests amenable to the routine clinical diagnostic laboratory. As the field of molecular diagnostics erupted, PCR quickly became commonplace in laboratory medicine [3
5].

6.1.2 Different Polymerase Chain Reaction Techniques—A Multitude of Opportunities for Companion Diagnostics In the modern laboratory, traditional PCR (first generation) had very limited applications due to, among others, issues involving burdensome workflow and accuracy. Following the introduction of heat-stable and more accurate polymerases alongside the development of RT detection (second-generation PCR), PCR is now commonly applied, in both the research and clinical lab, to detect a vast array of genetic alterations. PCR can be

COMPANION AND COMPLEMENTARY DIAGNOSTICS

114

6. POLYMERASE CHAIN REACTION

applied to detect structural DNA aberrations including translocations, deletions, inversions, and duplications (see later). In order to allow absolute quantification, digital PCR (dPCR) has been developed as the third generation of PCR technology. Before amplification the template is diluted to a certain concentration and dispersed to a number of microreaction units, which results in zero or one target DNA sequence(s) in each unit [6] (Fig. 6.2). After amplification the units containing copies of target DNA sequences show positive signals, whereas only background fluorescence is observed in the units with no target sequence. The Poisson distribution is then applied to quantify the mean number and fraction of positive units to reduce the errors generated by the presence of more than one copy of target sequence in some units. On this basis the initial copy number and concentration of target DNA can be obtained. The advent of the dPCR has elevated the detection of gene mutations to unprecedented levels of precision, especially in cancer-associated genes. dPCR has been utilized in the detection of tumor markers in cell-free DNA (cfDNA) samples from patients with different types of cancer in multiple sample types including plasma, cerebrospinal fluid, urine, and sputum, which confers significant value for dPCR in both clinical applications and basic research. dPCR is extensively used in detecting mutations in pathogens related to the typical features of infectious diseases (e.g., drug resistance) and mutation status of heteroplasmic mitochondrial DNA (mtDNA), which determines the manifestation and progression of mtDNA-related diseases as well as allows for the prenatal diagnosis of monogenic diseases and the assessment of the genome-editing effects. By increasing input volume, high sensitivity can be reached, that is for the detection of rare events, whereas increased numbers of partitions allows the detection of targets across a high dynamic range and rare mutation copy number variants. Compared with RT-PCR (qPCR) and sequencing, the higher sensitivity and accuracy of dPCR indicates a great advantage in the detection of rare mutations. However, as of today, dPCR has not been used in companion or complementary diagnostic applications to regulatory authorities. ing

10 8 8 7 6 0

10

5

5 Negative:

4

Positive:

T

# initial target molecules

9

PCR

3

u Co

2

er

Th

on n iss Po atio tim es

ng

nti

ycl

c mo

1

ing

ad

Lo

ts

le

mp

Sa

mp

Co

ut

do

en

a l re

m ar t

tica

Op

0

1

2

3

4

5

6

7

8

# positive PCR reactions

# targets = In[1-(pos/#wells)] x #wells

FIGURE 6.2 Principle of digital PCR. The patient sample is divided into multiple (e.g., thousands) reaction compartments (either well or droplets). Following thermal cycling and end point detection of targets using PCR the number of positive and negative compartments is determined. Using Poisson correction, the starting number of copies of the target can be calculated for absolute quantification. PCR, Polymerase chain reaction. Source: Courtesy Michael Zeder, Roche Diagnostics International AG, Rotkreuz, Switzerland.

COMPANION AND COMPLEMENTARY DIAGNOSTICS

6.1 POLYMERASE CHAIN REACTION TECHNOLOGY

115

6.1.3 Polymerase Chain Reaction Versus Other Diagnostic Technologies Current treatment strategies in oncology and other fields are rapidly changing. Targeted drugs and checkpoint inhibitors, that is, therapeutic antibodies, kinase inhibitors, and poly(ADP-ribose) polymerase inhibitors in most cases require a pretherapeutic companion or involve complementary diagnostic tests to identify the molecular alterations serving as targets for drugs. Defined and validated biomarkers, in most cases of genetic alterations, drive the efficacy of therapeutic drugs and therefore allow the identification of those patients likely or unlikely to benefit from a specific treatment. These analyses represent the basis of personalized or precision medicine. There are many examples for companion or complementary diagnostics in the treatment of malignancies (discussed below). Initially, almost all biomarkers were tissue based relying on immunohistochemistry (IHC), in situ hybridization, PCR, arrays, or sequencing. More recently, the detection of cfDNA in peripheral blood has been validated to provide relevant information to clinicians on genetic alterations for use in precision medicine [7]. cfDNA consists of short fragments of double-stranded DNA shed from tumors following necrosis or apoptosis during cell turnover and is characterized by unique somatic mutations that are not present in normal cells [8]. The discovery of cfDNA will increase the use of PCR and next-generation sequencing (NGS)-based companion and complementary diagnostics from the so-called liquid biopsies, whereas IHC-based solutions will continue to dominate companion and complimentary diagnostics in tumor tissue samples. PCR methods are popular due to ease of use, good sensitivity and specificity, timely turnaround for results, and relatively simple interpretation of data. PCR-based assays such as droplet dPCR, ARMS, and BEAMing are relatively cheap, highly sensitive and specific, but they can only detect a limited number of known mutations; PCR-based assays cannot easily detect copy number alterations and rearrangements (such as ALK or ROS1 fusions), tumor mutational burden (TMB), which are more easily detected via NGS [9]. In contrast, NGS-based assays such as TAM-Seq and CAPP-Seq are highly sensitive and specific and can detect multiple known and also unknown mutations; however, the technology is still costly and requires much longer turnaround and hands-on times. Recently, NGS-based CDx tests have been approved by regulatory authorities. In this regard, Foundation Medicine’s FoundationOne CDx (F1CDx) has been approved by the FDA using DNA isolated from formalin-fixed and paraffin-embedded (FFPE) tumor tissue specimens. In patients with advanced solid organ cancers the test detects the presence of genomic alterations in 324 cancer genes as a CDx to guide treatment with 17 therapeutics in patients with certain types of nonsmall cell lung cancer (NSCLC), melanoma, colorectal cancer, ovarian cancer, and breast cancer. Of interest, F1CDx has also been approved to report genomic biomarkers including microsatellite instability and TMB to assist in the selection of immune-checkpoint inhibitor therapy (see also “Chapter 8: Current NextGeneration Sequencing Based Companion Diagnostics and Their Analytical Validation” in this book). At present, about one in three technologies used for CDx are PCR based. (www.fda. gov/MedicalDevices/ProductsandMedicalProcedures1). Regardless of the technology applied to companion or complementary diagnostics, validation of these assays is critical for clinical and economic success [10
12]. We recently assessed the clinical and economic impact of inaccurate test results between

COMPANION AND COMPLEMENTARY DIAGNOSTICS

116

6. POLYMERASE CHAIN REACTION

laboratory-developed tests (LDTs) and an FDA-approved test (IVD) for the detection of epidermal growth factor receptor (EGFR) mutations using PCR [12]. Using a decision analytic model to estimate the probability of misclassification with LDTs compared to an IVD test, we estimated that in the best-case scenario 2.3% of newly diagnosed metastatic NSCLC patients would be misclassified with LDTs compared to 1% with an IVD test; an average of 477 and 194 progression-free life years were lost among the misclassified patients tested with LDTs compared to the IVD test, respectively. Costs associated with aggregate treatment for patients tested with LDTs were approximately $7.3 million higher than for patients tested with the IVD test due to higher drug and adverse event costs among patients incorrectly treated with targeted drugs or chemotherapy. These marked clinical and economic consequences to society are independent of the technology used and should push clinicians and laboratorians toward the utilization of diagnostic tests with demonstrated accuracy and clinical validation to maximize the potential of personalized medicine. In this regard, concerns over the generation of conflicting results (including falsenegative results) between DNA- and protein-based tests used to identify patients benefiting from new breakthrough treatment approaches have received attention not only among clinicians but also the FDA [13]. Thus due to ever increasing complexities in diagnostic technologies and clinical applications, the cooperation between clinicians, laboratorians, that is (molecular) pathologists, and regulatory authorities is critical to improve clinical drug selection and guide development and approval of new cancer gene therapies and molecularly targeted drugs.

6.2 COMPANION DIAGNOSTICS FOR ONCOLOGY APPLICATIONS CDx tests are important tools in practicing personalized/precision medicine. Most commonly, tests detecting a disease pathway associated biomarker(s) are paired with a specific drug(s) targeting that pathway. PCR approaches have been particularly successful in oncology where the analysis of somatic genetic alteration(s) informs treatment choices for many cancers such as BRAF mutation-positive melanoma, KRAS mutationnegative lung and colorectal cancer, EGFR mutation-positive lung cancer, BRCA mutation-positive breast and ovarian cancer, BCR-ABL1 mutation-positive chronic myeloid leukemia, and FLT3 mutation-positive acute myeloid leukemia. The majority of current PCR CDx tests are qualitative DNA assays for the detection of point mutations, indels, rearrangements or less frequently CDx measures quantitatively RNA transcript levels. In both cases CDx results target genes in pathways that are targetable by approved drugs (actionable genetic alterations) (Table 6.1). In current laboratory practice, CDx assays are either developed by a clinical laboratory (LDTs) or the laboratory adopts a test approved by regulatory authorities such as the FDA or carrying CE-mark approval (CE-IVD). Only a test that has received a CDx status by regulatory authorities has been validated at the level of an IVD for both the analytical and clinical claims; most importantly, the assay’s clinical utility (often referred to as clinical validity in the assay development process) has been proven in patients tested for the biomarker and treated with the associated drug in prospective clinical trials to demonstrate

COMPANION AND COMPLEMENTARY DIAGNOSTICS

6.2 COMPANION DIAGNOSTICS FOR ONCOLOGY APPLICATIONS

117

TABLE 6.1 FDA-Approved Companion Diagnostic Tests, Listed Chronologically by the Date of Original but Not Supplemental PMA.a Drug Name

Companion Device Name/Registration Date

Device Manufacturer

Zelboraf (vemurafenib)

cobas 4800 BRAF V600 Mutation Test, August 2011

Roche Molecular Systems, Inc.

Qualitative detection of BRAF V600E mutation in DNA from human melanoma tissue. Aid in selecting melanoma patients with tumors BRAF V600E positive for treatment with Zelboraf

Erbitux (cetuximab)b; Vectibix (panitumumab)b

therascreen KRAS RGQ PCR Kit, July 2012b, June 2014c

QIAGEN Manchester, Ltd.

Detection of seven somatic mutations in the human KRAS oncogene, in DNA from CRC tumor tissue. Aid in the identification of CRC patients for treatment with Erbitux and Vectibix based on a KRAS no mutation detected test result

Tarceva (erlotinib)b Tagrisso (osimertinib)c

cobas EGFR Mutation Test, May 2013b, November 2015c

Roche Molecular Systems, Inc.

Qualitative detection of exon 19 deletions, exon 21 (L858R) substitution mutations and resistance mutation T790M of the EGFR gene in DNA from human NSCLC tumor tissue. Aid in selecting patients with NSCLC for treatment with Tarceva-Exon 19 deletions and L858R or Tagrisso-T790M

Mekinist (tramatenib); Tafinlar (dabrafenib)

THxID BRAF Kit, May 2013

bioMe´rieux Inc.

Qualitative detection of the BRAF V600E and V600K mutations in DNA from human melanoma tissue. Aid in selecting melanoma patients with BRAF V600E-positive tumor for treatment with Tafinlar and as an aid in selecting melanoma patients with BRAF V600E or V600K-positive tumor for treatment with Mekinist

Gilotrif (afatinib)b Iressa (gefitinib)c

therascreen EGFR RGQ PCR Kit, July 2013b, July 2015c

QIAGEN Manchester, Ltd.

Qualitative detection of exon 19 deletions and exon 21 (L858R) substitution mutations of the EGFR gene in NSCLC tumor tissue. Aid to select patients with NSCLC for whom Gilotrif or Iressa, EGFR TKI are indicated

Lynparza (olaparib) or Zejula (niraparib)

BRACAnalysis CDx, December 2014a, January 2018b

Myriad Genetic Laboratories, Inc.

Qualitative detection of variants in the protein coding regions and intron/exon of the BRCA1 and BRCA2 genes in DNA from whole blood by PCR, Sanger sequencing and multiplex PCR. Aid in selecting BRCA 1 or 2 in breast and ovarian positive patients for treatment with Lynparza or Zejula maintenance therapy. This assay is to be performed only at Myriad Genetic Laboratories

Erbitux (cetuximab); Vectibix (panitumumab)

The cobas KRAS Mutation Test, May 2015

Roche Molecular Systems, Inc.

Detection of seven somatic mutations in codons 12 and 13 of the KRAS gene in DNA from human CRC tumor tissue. Aid in identification of CRC patients for whom treatment with Erbitux or with Vectibix may be indicated based on a no mutation detected result

Summary of Intended Use

(Continued)

COMPANION AND COMPLEMENTARY DIAGNOSTICS

118

6. POLYMERASE CHAIN REACTION

TABLE 6.1 (Continued) Drug Name

Companion Device Name/Registration Date

Device Manufacturer

Summary of Intended Use

Imatinib mesylate

KIT D816V Mutation Detection by ARUP PCR for Gleevec Eligibility in Laboratories, ASM, December 2015 Inc.

Qualitative detection of KIT D816V mutational status from fresh bone marrow samples of patients with aggressive systemic mastocytosis. Aid in selection of ASM patients for treatment with Gleevec. This assay is for professional use only and is to be performed at a single laboratory site

Tarceva (erlotinib)b Tagrisso (osimertinib)c

cobas EGFR Mutation Test v2, June 2016b, September 2016c

Roche Molecular Systems, Inc.

Qualitative detection of defined mutations of the EGFR gene in DNA from NSCLC patients in tumor tissue or cfDNA from EDTA plasma. Aid in selecting NSCLC patients for treatment with Tarceva-Exon 19 deletions and L858R or Tagrisso-T790M Patients who are negative for these mutations should be reflexed to routine biopsy and testing for EGFR mutations with the tumor tissue

Rydapt (midostaurin)

LeukoStrat CDx FLT3 Mutation Assay, April 2017

Invivoscribe Technologies, Inc.

Detection of ITD mutations D835 and I836 in the FLT3 gene in DNA from mononuclear cells from peripheral blood or bone marrow aspirates of patients diagnosed with acute myeloid leukemia (AML). Aid in the selection of patients with AML for treatment with RYDAPT

Idhifa (enasidenib) Abbott RealTimeIDH2, August 2017

Abbott Molecular, Inc.

Qualitative detection of SNVs coding nine IDH2 mutations (R140Q, R140L, R140G, R140W, R172K, R172M, R172G, R172S, and R172W) in DNA from human blood (EDTA) or bone marrow (EDTA). Aid in identifying AML patients with an IDH2 mutation for treatment with IDHIFA

Tasigna (nilotinib)

MolecularMD Corporation

Quantitative detection of BCR-ABL1 transcripts (e13a2/b2a2 and/or e14a2/b3a2) in mRNA in peripheral blood specimens from chronic myeloid leukemia (CML) t(9:22) positive patients. Aid in monitoring BCR-ABL mRNA transcript levels in t(9;22) positive CML under treatment with TKIs and also as an aid in identifying chronic CML patients being treated with Tasigma who may be candidates for treatment discontinuation and for monitoring of treatment-free remission

MolecularMD MRDx BCR-ABL Test, December 2017

a

US Food and Drug Administration, https://www.fda.gov/MedicalDevices/ProductsandMedicalProcedures. Original PMA date. Supplemental PMA date, type of PMA submitted if extension to the original PMA label is requested.

b c

ASM, Aggressive systemic mastocytosis; CDx, companion diagnostic; cfDNA, circulating-free tumor cell-free DNA; CRC, colorectal cancer; EGFR, epidermal growth factor receptor; IDH2, isocitrate dehydrogenase-2; ITD, internal tandem duplication; NSCLC, nonsmall cell lung cancer; PCR, polymerase chain reaction; SNVs, single-nucleotide variants; TKI, tyrosine kinase inhibitors; PMA, premarket approval; EDTA, Ethylenediaminetetraacetic acid; CML, Chronic myeloid leukemia.

COMPANION AND COMPLEMENTARY DIAGNOSTICS

6.2 COMPANION DIAGNOSTICS FOR ONCOLOGY APPLICATIONS

119

improvements in health outcomes in the context of efficacy and safety. The first concurrent approval for a PCR-based CDx and a drug took place in 2011 for the cobas BRAF Mutation Test and vemurafenib (Zelboraf) for metastatic melanoma. The clinical study that led to approval of the test and drug was the NO25026 clinical trial (BRIM3), an international, randomized, open-label, controlled, multicenter, Phase III study to evaluate the efficacy of vemurafenib versus dacarbazine in previously untreated patients with unresectable stage IIIC or stage IV melanoma with a V600E BRAF mutation (as measured by the cobas BRAF Mutation Test). The response rate in the vemurafenib arm was 48.4% compared to 5.5% in the dacarbazine arm, confirming the predictive value of the BRAF test in patients with melanoma who harbor a V600E mutation [14]. Other examples include the therascreen KRAS RGQ PCR test approved as a CDx for cetuximab (an EGFR-directed monoclonal antibody) based on retrospective results of the CA225025 clinical trial that compared cetuximab to best supportive care (BSC) in patients with metastatic colon cancer [15]. For the KRAS mutation-negative [wild-type (WT)] population the median overall survival was 8.6 months in the cetuximab group versus 5.0 months in the BSC group. There was no difference in overall survival for the KRAS mutation-positive population between cetuximab and BSC (4.8 vs 4.6 months). This demonstrated that a mutation in codon 12 or 13 of the KRAS gene was a negative predictor of response to cetuximab. The cobas EGFR Mutation Test is approved as a CDx for the EGFR tyrosine kinase inhibitors (TKIs) erlotinib and osimertinib in patients with NSCLC and activating mutations in the EGFR gene. Approval for erlotinib was obtained by retrospective testing of patients in the EURTAC clinical trial comparing erlotinib versus chemotherapy in patients with an exon 19 deletion or exon 20 L858R substitution mutation as determined by a clinical trial assay (CTA). In a cohort of patients with available samples from the study, among those patients with the above mutations as identified by the cobas EGFR test, the progressionfree survival (PFS) was 10.4 months in the erlotinib arm versus 5.4 months in the chemotherapy arm [16]. Approval for osimertinib, a so-called third-generation EGFR TKI, was obtained with results from the AURA2 trial which was a single-arm clinical trial of osimertinib in patients with NSCLC patients who had disease progression after prior EGFR TKI therapy and whose tumor harbored an EGFR T790M mutation as detected by the cobas EGFR Mutation Test. The T790M mutation is resistant to first- and second-generation EGFR TKI therapy (erlotinib, gefitinib, and afatinib), but sensitive to osimertinib. There was a 62.3% response rate to osimertinib in this trial. Based on this response rate, osimertinib was approved for use in this group of NSCLC patients by the FDA and the cobas EGFR Mutation Test v2 was approved as a CDx [17]. Current molecular testing guidelines for treatment with targeted therapeutics require testing for cancer mutations at the time of initial diagnosis to inform personalized treatment for a particular cancer type [18,19]. These recommendations are issued by professional societies, for example, College of American Pathologists, the International Association for the Study of Lung Cancer, the Association for Molecular Pathology, Society for Clinical Pathology, American Society of Clinical Oncology, and European Society of Medicinal Oncology. PCR-based CDx approved in the last decade are listed in Table 6.1.

COMPANION AND COMPLEMENTARY DIAGNOSTICS

120

6. POLYMERASE CHAIN REACTION

6.3 DEVELOPMENT PROCESS 6.3.1 General Considerations In the development of FDA-approved or CE-mark IVDs, it is important to follow an organized and well-thought-out process to ensure ultimate success for the final assay product. It is also important to engage with regulatory bodies early in the planning stage to aide in the development process. An IVD level assay will ultimately need to demonstrate verifiable accuracy and clinical validity. A project generally begins with an analysis phase which is intended to generate data on whether a specific product concept is feasible and potentially commercially marketable. Activities here often include preliminary experiments to show the technical possibility of a specific technology or assay design. An initial medical value assessment of the intended product is conducted to explore the intended uses, assess the clinical usefulness of the assay, and to outline possible approaches to clinical validation. Preliminary marketing data, health economic evaluation, and customer requirements are assembled to ascertain the potential economic impact of the intended product. Essentially, this phase is to identify a promising assay and find out what is needed to move the project forward, that is, define the goals of the assay design that will be needed to make it successful. The next phase is typically a feasibility phase which is intended to determine if the new product is technically feasible and marketable. It includes the assessment of alternative technical solutions and the transfer of design from a research level to a development level where the goal is to optimize the assay for the anticipated market. The primary intended use and the associated health economic impact are determined, and the clinical validation is planned. By the end of this phase, it has been determined what is to be made, and the best feasible concept to do so has been identified. The third phase of this design control process is assay development. Reagent and software components, as well as process and testing methods are optimized and finalized. Design verification activities are initiated, as applicable, such as technical performance verification testing, stability testing, and software level verification. Test methods and specifications are developed, transferred, and validated using statistical methods where applicable. Instructions for manufacturing reagents and system components (e.g., protoand pilot instruments) are developed, and process capabilities are analyzed. Upon completion of the development phase the product should meet current product requirements. If the product does not meet one or more product requirements, a risk analysis (including business, technical and financial assessments) with potential mitigations should be performed to determine the merit of registering and launching the product with its current performance characteristics. The last phase of product development is where product and process validation activities are completed. In addition, performance of the product produced in a manufacturing facility shall be supported by internal and/or external validation studies. Upon completion of these validation activities, results should be assessed during a design review to ensure the product meets technical, clinical, and customer needs. The necessary filings will be reviewed by the regulatory authorities, and approval will be given so the product can be launched and commercially distributed.

COMPANION AND COMPLEMENTARY DIAGNOSTICS

6.3 DEVELOPMENT PROCESS

121

This process may become more complicated when developing a CDx in collaboration with a pharmaceutical partner. In this situation the pharmaceutical company may require a test as part of the screening for enrollment into a clinical trial. An assay is therefore often needed prior to the clinical validation of the test; indeed, the clinical trial in this case will provide proof of the clinical utility of the assay which would be considered an investigational device for research use only until such validation. As such, it may be necessary to develop a prototype assay for the clinical trial (a CTA) that has not been clinically validated. In such a case, it is then necessary to bridge from the CTA to the IVD assay once the results of the clinical trial are known to provide proof of the clinical utility required for regulatory approval.

6.3.2 Analytical Verification and Validation As part of the assay development, a number of technical performance verification studies must be performed to confirm the fitness of the assay. These are crucial for regulatory approval. Assistance in determining and performing verification studies can be found in references from the Clinical Laboratory Standards Institute (CLSI) and the International Organization for Standardization. The list of verification studies (Table 6.2) can be contracted or expanded depending upon the assay under development, usually in consultation with regulatory agencies. For the cobas EGFR Mutation Test the Limit of the Blank was established by testing NSCLC specimens which were WT (nonmutated) for the EGFR gene, which in this case was determined to be zero for all mutations. In the case of plasma, healthy donor (HD) plasma which was WT for EGFR was also tested and found to be zero for all mutations. Of note, the cobas EGFR Mutation Test detects EGFR mutations in exon 18 (G719X) substitutions, exon 19 deletions, exon 20 insertions and substitutions (T790M, S768I), and exon 21 substitutions (L858R, L861Q) along with one internal control. The Limit of Detection (LoD) is established by testing the target sequence at different concentration levels. The LoD is set at that level at which the assay correctly identifies 95% TABLE 6.2 Verification Studies Often Needed for Approval of Companion Diagnostic Tests. LoD: predominant mutations

Reagent lot interchangeability

LoD verification of rare mutations

Cross contamination

Potentially interfering endogenous substances

Prepared specimen stability

Potentially interfering exogenous substances

Activated MMX stability

Potentially interfering microorganisms

Open container stability

Robustness (guardband results)

Plasma specimen handling stability

Reagent kit stability plasma

Contrived sample comparison

Exclusivity LoD, Limit of detection; MMX, MasterMix.

COMPANION AND COMPLEMENTARY DIAGNOSTICS

122

6. POLYMERASE CHAIN REACTION

of the samples tested. In the cobas EGFR test, FFPE tissue (FFPET) DNA extracts for mutations in exon 18, 19, 20, and 21 were blended with WT EGFR DNA to target mutation levels of 1.25%
10% (determined by an NGS method). Eight replicates for each DNA mutation was tested by each of three cobas EGFR test kits or 24 replicates per mutation at four different concentration levels. In total, 31 mutations were evaluated. The study demonstrated that the cobas EGFR test could detect mutations in EGFR exons 18, 19, 20, and 21 with at least a 5% mutation level using the standard input of 50 ng DNA. The situation for plasma was more complicated. There was concern by regulatory authorities that there may be differences in assay performance in plasma from NSCLC patients as compared to HD plasma. A full LoD assessment was not possible with NSCLC patient plasma due to scarcity and difficulty in obtaining the large volumes that would be needed for a full set of preclinical studies. In collaboration with the regulatory authorities, it was agreed that a commutability study of major mutations between NSCLC patient and HD plasma would be acceptable if there were no differences. The study of sheared cell line DNA diluted in HD plasma or NSCLC plasma yielded equivalent results at concentrations near LoD. Finally, sheared cell line DNA containing each of the seven mutation classes detected by the cobas EGFR test was added to HD plasma that was WT for EGFR. Serial dilutions were prepared, and 24 replicates of each panel member were tested, using each of three cobas EGFR test kit lots. Cell line DNA was mechanically sheared to an average size of 220 bp and diluted into a WT DNA background of approximately 100,000 copies/mL. The study demonstrated that the cobas EGFR test could detect mutations in EGFR exons 18, 19, 20, and 21 with # 100 copies of mutant DNA per mL of plasma using the standard input of 25 µL of DNA stock, which represents a detection level of approximately 0.1% mutant DNA. Potentially interfering substances must be assessed and are divided into endogenous and exogenous substances. The CLSI manuals can be helpful in deciding which substances should be tested and at what concentrations. This is often based on the source of the sample to be tested. For the cobas EGFR test the endogenous substances triglycerides and hemoglobin were shown not to interfere with the cobas EGFR test when the potential interfering substance was added to the lysis step during the specimen preparation procedure. Exogenous substances tested included drugs which were felt to be common to patients with chronic lung disease. Albuterol, ipratropium, and fluticasone did not interfere, nor did the antibiotics ceftazidime, imipenem
cilastatin, piperacillin
tazobactam and cilastatin, as well as betadine and lidocaine which might be used during a lung biopsy. Potentially interfering lung pathogens Streptococcus pneumoniae and Haemophilus influenzae were found not to interfere with the cobas EGFR test when added to specimens containing WT and mutant EGFR sequences during the tissue lysis step. The potentially interfering endogenous substances tested during the development of the plasma test which did not interfere included triglycerides, bilirubin, and hemoglobin up to 1.5 g/L. However, hemoglobin at 2 g/L did interfere, and albumin at 60 g/L may have interfered. No new exogenous substances were tested. One additional microorganism was tested, Staphylococcus epidermidis, which is a common skin contaminant which did not interfere with the assay. Exclusivity of the assay must also be determined by testing known DNA sequences which might cross-react with an assay. In the case of the cobas EGFR test the specificity

COMPANION AND COMPLEMENTARY DIAGNOSTICS

6.3 DEVELOPMENT PROCESS

123

was evaluated by testing lung-related microorganisms and plasmids containing the sequences from each of the HER2, HER3, and HER4 genetic regions analogous to the sequences in EGFR exons 18, 19, 20, and 21 amplified by the cobas EGFR test. Structurally related epidermal receptor tyrosine kinase protein analog sequences (EGFR/HER1, HER2, HER3, and HER4) were shown not to cross-react with the cobas EGFR test when the potential cross-reactive sequence was added at an appropriate DNA input level to the isolated DNA stock prior to the amplification/detection procedure. Results indicated that the observed mutations for all tested FFPET specimens matched the expected mutation, as determined by sequencing, in the presence and absence of the added HER gene plasmid DNA. However, there was one mutation, the EGFR exon 19 mutation L747S which was tested and shown to cross-react with the cobas EGFR test. Potentially interfering lung microorganisms S. pneumoniae and H. influenzae in tissue and the skin microorganism S. epidermidis in plasma were found not to cross-react with the assay. Repeatability is generally an internal process required to regulatory authorities to ensure that the assay produces the same results over more than one assay kit lot by more than one operator. The FDA has guidelines regarding this process and for most assays, a minimum of two replicates per analyte using two different reagent lots and two assay analyzers by two operators over multiple nonconsecutive days is required. For the cobas EGFR test in tissue, two repeatability tests were performed. The first included two WT FFPET samples and four FFPET specimen samples covering six EGFR mutations. The second included one WT and three EGFR mutation FFPET samples covering rarer mutations. The first had a correct call rate of 97% and the second 99%. Reproducibility is a way to evaluate and emulate the assay under real-world conditions. The studies are generally performed by the external laboratories which are familiar with the technique. As above, the FDA has provided guidance on reproducibility studies. In addition the coefficient of variation (CV), an important measure of assay variability, both within a single laboratory and between laboratories as well as within a single lot of reagents and between lots, can be calculated from these studies. For molecular tests such as the cobas EGFR test, a typical scheme involves three testing sites which will test the samples in duplicate with three reagent lots over 5 nonconsecutive days. For the EGFR test in tissue, two studies were performed, one with multiple samples from FFPET samples of the predominant EGFR mutations exon 19 deletion and the L858R substitution mutation at two mutation allele frequencies, 5% and 10%, and a second study of less common mutations (exon 18 G719X, exon 20 T790M, exon 20 S768I, exon 20 insertion, and exon 21 L861Q) at two levels, LoD and 2X LoD. WT samples were included in both studies. The results in both studies were excellent, and importantly the results for the WT were all correct showing high specificity. The CV in both studies for mutation panel members was less than 9.2% and for the WT less than 1.3%. A similar study was performed in plasma. A panel of seven mutations was prepared in a four-member panel at 100 copies/ mL (LoD) and 300 copies/mL (3 3 LoD) along with one WT sample. The overall agreement was excellent for the mutation panel members with only one mutation less than 97.2% and 100% agreement for the WT sample. Taken together, the repeatability and reproducibility studies highlighted the robustness of the cobas EGFR Mutation Test which is crucial when preparing an IVD level assay or a CDx.

COMPANION AND COMPLEMENTARY DIAGNOSTICS

124

6. POLYMERASE CHAIN REACTION

6.3.3 Clinical Validation The final requirement for an IVD level assay or a CDx is the demonstration of clinical utility using clinical samples, preferably from a prospective clinical trial. The clinical trial confirms that the assay is prognostic or predictive for response to a therapeutic agent. It is not enough to show that the assay is closely correlated with clinical benefit, but a method comparison study is required to prove that the assay is actually measuring what it claims. Furthermore, it is necessary to have clinical samples from both the screen positive (patients tested and included in the drug trial) and screen negative (patients tested but not included in the drug trial) populations as it is important to be sure that the assay is not missing patients with the target mutation. Therefore it is important to work in close collaboration with the pharmaceutical partner and regulatory agencies to collect the samples and data necessary to confirm the clinical benefit. This includes more than just tissue or plasma samples, but also demographic data and other potential covariates, to confirm the assay is being used in the correct intended use population. When testing a multianalyte assay, all analytes must be detected in the clinical trial and also demonstrate clinical benefit. Only those analytes which demonstrate clinical validation will be included as part of the IVD label. The cobas EGFR Mutation Test is instructive in this situation. The original approval for the assay in tissue was based on the retrospective analysis of the EURTAC clinical trial which was a Phase III, multicenter, open-label, randomized study of erlotinib versus standard of care platinum doublet chemotherapy as first-line therapy in chemotherapy-naı¨ve patients with advanced NSCLC whose tumors harbored EGFR exon 19 deletions or exon 21 (L858R) substitution mutations, as assessed by a CTA [16]. Tissue samples from the trial were retrospectively tested with cobas EGFR test. This analysis showed that those patients treated with erlotinib had an improved PFS of 10.4 months compared to 5.4 months in the chemotherapy arm. Based on this clinical trial, erlotinib was approved by the FDA for use in patients with activating mutations in EGFR. Even though the cobas EGFR test detects seven major mutations in EGFR, it only received CDx designation for treatment with erlotinib for exon 19 deletions and L858R substitution mutations as they were the only mutations included in the clinical trial. However, based on the technical performance studies listed above, the other mutations did receive an analytical designation by the FDA. To expand the mutations detected by the cobas EGFR test required clinical validation in another clinical trial. The AURA2 trial was a Phase II, multicenter, open-label, single-arm study, assessing the safety and efficacy of osimertinib as a second-line or greater than or equal to third-line therapy in patients with advanced NSCLC, who had progressed following prior therapy with an approved EGFR TKI agent. All patients were required to have EGFR T790M mutation-positive NSCLC as detected by the cobas EGFR test [17]. In this trial, patients with a T790M mutation had an objective response rate of 62.3% and resulted in FDA approval for osimertinib for the second-line treatment of NSCLC patients who had progressed on a first-line EGFR TKI and now were T790M mutation positive; along with the drug approval, the cobas EGFR test expanded its CDx designation to include exon 19 deletions, L858R, and now T790M mutations. This study also presented an additional problem for the cobas EGFR Mutation Test. Patients enrolled on this study had been tested with the original version of the cobas test,

COMPANION AND COMPLEMENTARY DIAGNOSTICS

6.3 DEVELOPMENT PROCESS

125

but while this clinical trial was ongoing, an additional mutation (L861Q) was included in the assay. Due to this change, it was necessary to retest the tissue samples from all of the eligible patients in the study with the new version of the test which demonstrated a 97% concordance between the original version and the new version of the assay. In essence, this was a bridging study between the two versions of the assay. In the end, not only did the cobas EGFR test get an expanded CDx claim but also approval by the FDA of the newer version of the assay. The approval of the cobas EGFR test in plasma was more complicated than for tissue and required close communication with the FDA. The ENSURE study was a multicenter, open-label, randomized Phase III study to evaluate the efficacy and safety of erlotinib versus gemcitabine/cisplatin as the first-line treatment for stage IIIB/IV NSCLC patients with exon 19 deletion or L858R substitution mutations in the tyrosine kinase domain of EGFR [20]. During the course of this trial, plasma samples were collected prospectively at the screening visit. While there was sufficient plasma for the cobas EGFR test, there was not enough plasma for analytical accuracy studies. However, there were plasma samples available from other studies with a similar intend to treat population. ASPIRATION was the primary source of tissue EGFR mutation-positive plasma samples, and these were supplemented with tissue EGFR mutation-negative plasma samples from other lung cancer studies [21]. A sensitivity analysis was initially performed to ensure there were no substantive differences between the population of patients in ENSURE and the populations in the augmented ASPIRATION cohort. The positive percent agreement (PPA) was 87.5% between the cobas EGFR test and NGS in plasma. All of the NGS mutation-positive, cobas EGFR mutation-negative samples had a mutation percentage below the LoD of the cobas test. The negative percent agreement (NPA) between the cobas test and NGS was 96.8%. The PFS for those patients in the ENSURE trial who were plasma mutation positive was 11 months for erlotinib compared to 5.6 months for chemotherapy. These results were essentially identical to the results for tissue EGFR mutation-positive patients (11.1 vs 5.7 months) thereby confirming the clinical validity of the cobas EGFR test in plasma. The cobas EGFR assay became the first plasma-based molecular diagnostic to gain CDx designation by the FDA. As with EURTAC, due to the clinical trial limiting enrollment to patients with exon 19 deletions or the L858R substitution mutation, only these mutations received CDx designation in combination with erlotinib. Similar to the cobas EGFR test in tissue noted above, the AURA2 trial was used to expand the CDx claim in plasma. In AURA2, plasma was collected at the initial screening visit and tested with the cobas EGFR test. The objective repose rate (ORR) for patients with a T790M mutation-positive result by both tissue and plasma samples was 61.5%, which was essentially identical to the 61% observed ORR in the tissue mutation-positive population. With the clinical validity confirmed, the T790M mutation in plasma was given CDx designation for osimertinib in NSCLC patients.

6.3.4 Considerations for the Statistical Analysis of Companion Diagnostics Clinical Studies Statistical analysis to demonstrate the clinical validity of a CDx product depends on the design of the clinical studies. When the need for the CDx biomarker is identified in the early

COMPANION AND COMPLEMENTARY DIAGNOSTICS

126

6. POLYMERASE CHAIN REACTION

development phase of the therapeutic product, two possibilities exist (1) an analytically validated final version of the CDx test is available. Let us call this the IVD test assay, or (2) a prototype or the research version of the CDx test is available. Let us call this the CTA. The analysis to demonstrate the clinical validity of the assay depends on which version of the test is used to select the patients for the pivotal clinical trial. If the final IVD version of the test is used to select patients, the statistical analysis to demonstrate the clinical validity of the IVD test simply requires demonstrating analytical accuracy of the test. If the CTA version of the test is used to select the patients, it is required to demonstrate that the performance characteristics of the IVD version of the test are very similar to the CTA test. This is known as a bridging study. The statistical analysis to demonstrate the clinical validity of the IVD test requires demonstrating drug efficacy for patients positive by the IVD version of test in addition to demonstrating analytical accuracy of the test. 6.3.4.1 Statistical Analysis When In Vitro Diagnostic Version of the Co-Dx Is Available for the Pivotal Clinical Trial To select the patients for the pivotal clinical trial the IVD test can be prospectively used. For this study design the statistical analysis for the IVD test simply requires demonstrating analytical accuracy of the test in the intended use population for the safe and effective use of the therapeutic product. Ideally, banked samples from all of the patients screened in the pivotal clinical trial are tested by a sequencing method. However, if the number of screened patients is very large, then it is usually acceptable to retest a subset of the patient samples. The selected subset ideally includes samples from all the test-positive patients and a subset of test-negative patients from the screened population. To demonstrate the accuracy of the test the results from the IVD test are compared with the results from a DNA sequencing method as shown in Table 6.3. Performance of the IVD test can be characterized by the following unadjusted agreement measures: a a1c d NPA1 5 b1d PPA1 5

where PPA1 refers to the positive percent agreement of the IVD test with the sequencing method, and NPA1 refers to the negative percent agreement of the IVD test with the TABLE 6.3

Comparison of Results of Sequencing Method and IVD Test. Sequencing Method Results

IVD test results

Positive

Negative

Not Sequenced

Total

Positive

a

b

e

a1b1e

Negative

c

d

f

c1d1f

Total

a1c

b1d

e1f

N

Samples in cell “e” are not sequenced due to unavailability or by design, and test-negative patients (c 1 d) are randomly selected to tested by a sequencing method. IVD, In vitro diagnostic.

COMPANION AND COMPLEMENTARY DIAGNOSTICS

127

6.3 DEVELOPMENT PROCESS

sequencing method. The corresponding two-sided 95% CI can be calculated using Wilson score intervals [23]. Unbiased estimates of PPA and NPA are obtained by adjusting for the samples not selected for sequencing as follows: ða=a 1 bÞ 3 ða 1 b 1 eÞ ða=a 1 bÞ 3 ða 1 b 1 cÞ 1 ðc=c 1 dÞ 3 ðc 1 d 1 fÞ ðd=c 1 dÞ 3 ðc 1 d 1 fÞ NPA2 5 ðd=c 1 dÞ 3 ðc 1 d 1 fÞ 1 ðb=a 1 bÞ 3 ða 1 b 1 eÞ PPA2 5

To obtain the unbiased estimates of PPA and NPA an assumption is made that the samples not sequenced are substantially similar to the samples that are sequenced. This assumption is verified by comparing the demographics, sample characteristics, and relevant medical history of the patients whose samples are sequenced versus not sequenced to demonstrate that the estimates of PPA2 and NPA2 are representative of the intended use population. 6.3.4.2 Bridging Study: When Clinical Trial Assay Is Utilized to Select Patients for the Pivotal Clinical Trial If an analytically validated final version of the test is not available before the beginning of the pivotal clinical trial, a prototype version of the CDx test (also called the CTA) is used to screen the patients. Banked samples from all the patients screened for the trial are retested by the final IVD version of the test to demonstrate the clinical validity of the IVD test. Results from the IVD version of the test are compared with the CTA version of the test, and the agreement between the two versions of the test is calculated. This type of study design is called a bridging study and requires additional analyses for demonstrating the clinical validity of the IVD version of the test. An agreement between the CTA version and the IVD version of the test is evaluated by comparing the results as shown in Table 6.4. Statistical analysis for this study design requires the calculations of PPA and NPA to estimate the agreement between CTA and IVD version of the test. Note that CTApositive patients (a 1 c) are enrolled in the study; however, to clinically validate the IVD test, it is required to demonstrate that the drug is effective for patients positive by IVD test that is, a 1 b patients. To accomplish this, a two-step approach is followed. First, the drug efficacy is evaluated and for patients positive by both versions, that is, patients in cell “a.” Next, a sensitivity analysis is performed to show efficacy for patients in cells “a” and “b.” TABLE 6.4 Comparison of Results of a CTA and the IVD Test. CTA Results

IVD test results

Positive

Negative

Total

Positive

a

b

a1b

Negative

c

d

c1d

Total

a1c

b1d

N

CTA, Clinical trial assay; IVD, in vitro diagnostic.

COMPANION AND COMPLEMENTARY DIAGNOSTICS

128

6. POLYMERASE CHAIN REACTION

This approach is described in detail in [22], see also “Principles for Codevelopment of an In Vitro Companion Diagnostic Device with a Therapeutic Product-Draft Guidance for Industry and Food and Drug Administration Staff,” https://www.fda.gov/ MedicalDevices/ProductsandMedicalProcedures/InVitroDiagnostics/ucm407297.htm.

6.4 CLINICAL STUDY CONDUCT Of the utmost importance, clinical studies for a CDx require close and mutual collaboration between the pharmaceutical (pharma) and diagnostic partners. This simple, yet essential statement belies a shift in thinking for both parties. Although each of them is familiar with the requirements for and logistics of conducting trials separately to obtain claims for their respective products, both parties have a stake in a CDx and must pool their strengths. Both parties must be transparent with each other, recognize the expertise of their respective clinical operations teams, and align on the items listed in Table 6.5. The strength of the pharma partner lies in the interaction with clinical sites, that is, patient enrollment and treatment interventions. The diagnostic partner, in contrast, has broad expertise in interaction with laboratories, that is, sample handling and testing. The expertise in these respective areas and the broader objectives of the pharma and diagnostic partners makes it necessary to have two separate clinical study protocols, one sponsored by pharma, and the other sponsored by the diagnostic partner. It is important to understand that success of a CDx is not only measured by regulatory claims but also by the successful and long-term business relationship between the pharmaceutical and diagnostic companies. Furthermore, it is highly recommended that the partners planning a trial for CDx work together with the regulatory agencies such as the FDA. The regulation of drugs is performed by one group, in the United States called CDER (Center for Drug Evaluation and Research) while laboratory tests are regulated by CDRH (Center for Devices and Radiologic Health). These two groups must also act collaboratively to ensure a smooth and coordinated approval process for both the drug and assay. Other regions or countries have their own regulatory agencies which may have slightly different approval pathways. Engaging with regulatory agencies early for assistance in designing the required technical TABLE 6.5 Areas of Alignment Between the Diagnostic and Pharma Partners. Diagnostic and Pharma Proposed Intended Use for the CDx Diagnostic and pharma clinical study protocols Informed consent form Laboratory manual Diagnostic and pharma clinical study databases Diagnostic and pharma regulatory submission strategy and timeline Diagnostic and pharma commercialization strategy for the CDx CDx, companion diagnostic.

COMPANION AND COMPLEMENTARY DIAGNOSTICS

6.4 CLINICAL STUDY CONDUCT

129

performance studies and clinical trials is often critical in providing the best chances for ultimate approval. A CDx can be achieved by employing either a prospective or retrospective study design, with the agreement of the regulatory agency. In a prospective clinical study a diagnostic test is used to screen patient samples for selection or stratification into a drug study. In a retrospective clinical study the diagnostic is used to test samples that were collected and banked by the pharmaceutical partner in the course of a clinical trial. In both types of studies, guidance for sample collection and storage are provided to the clinical investigators in a laboratory manual. The disadvantage of a retrospective clinical study commonly is that the diagnostic partner may not have input to the creation of the laboratory manual that indicates the critical aspects of sample and data collection, shipment and testing of samples. For example, if the diagnostic partner was not involved, the samples may have been collected by processes not validated by the diagnostic partner. In addition, if the diagnostic partner was not involved in the previous data collection, sample processing details and data from screen-failed patients may not be available for inclusion in the CDx. It should be noted that both the diagnostic test and the pharmaceutical drug are considered investigational since neither have been approved for the proposed intended use. Regardless of study design and whether the study is to support a CDx, a clinical project timeline is fundamentally defined by five phases (Fig. 6.3): planning, preparation, study execution, analysis, and postanalysis. During the planning phase of a prospective or retrospective clinical study, all pharmaceutical and diagnostic team members should be identified with their roles and responsibilities clearly delineated. This team roster should be maintained and made available to all members to facilitate effective and efficient communications. A common cause of confusion or delay in the project timeline is due to misunderstanding as to who are the key decision makers. The team members should agree early on the expectations of all parties. As applicable, these include the clinical operations teams; clinical research organization (CRO); investigational testing sites; clinical investigators; patients (commitment of patients to comply to study procedures, complete subject diaries or return for follow-up); data management teams; and biostatisticians. Both the diagnostic and pharma partners will also have support from the clinical science team

FIGURE 6.3 Key activities during five phases of a clinical study.

COMPANION AND COMPLEMENTARY DIAGNOSTICS

130

6. POLYMERASE CHAIN REACTION

(responsible for trial design) and other parties with in-depth knowledge on the product (drug or CDx test). It is recommended that the CDx be managed by two separate clinical protocols that would each require review and approval by an independent review board (IRB) or independent ethics committee (IEC). The strategy describing the studies that would support the proposed intended use should be stated in a clinical validation plan. As the sponsor of the investigational drug study, the pharmaceutical partner ensures that their protocol indicates the purpose of the clinical trial, proposed intended use, inclusion and exclusion criteria, study design, number of patients to be screened and enrolled, and the duration of the study. The pharmaceutical protocol must also indicate the investigational status of the diagnostic test and specify how the results of the test will be used in the drug study. As the sponsor of the diagnostic study, the diagnostic protocol must align with the pharmaceutical study (in other words, the pivotal trial for registration purposes) and indicate the clinical validity objective. A benefit of having two protocols is that each sponsor can include secondary and exploratory objectives in their respective protocols that are independent of the CDx. While the clinical investigators adhere to the drug protocol, the investigational testing sites should adhere to the diagnostic protocol. This leads to the necessity of maintaining two separate databases, one managed by each partner. Similar to the two separate protocols, the diagnostic and pharma databases must align. Both databases should have the same clinical characteristics (e.g., demography) and clinical outcome information. In addition, it is important for both partners to be aware of protocol deviations that led either party to invalidate a patient’s data or sample result since this information may affect analysis. A common point of confusion during the CDx collaboration occurs between the diagnostic and pharma partners when defining screen failures and the data collected from these patients. Patients are considered a screen failure if the diagnostic test generates a result that may prohibit the patient from being enrolled in the pharma study. Since the demographics and clinical characteristics for patients in this subset may not be used by pharma as they assess their intent to treat population, pharma may not see the benefit in providing the data from this subset to the diagnostic partner. It is critical for the pharma team to recognize, however, that this information is mandatory for the diagnostic partner since the screen failures are included in the intent to diagnose population. The informed consent form (ICF) is developed by the pharmaceutical clinical operations team to align with the drug study protocol. The ICF should be reviewed by the diagnostic clinical operations team to verify that it includes the mandatory information required by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Harmonized Tripartite Guideline for Good Clinical Practice E6 (https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/ Guidelines/Efficacy/E6/E6_R1_Guideline.pdf) and, specifically, at least four key elements: one, a statement that the test is investigational for the proposed intended use; two, the diagnostic partner has access to patient samples and data to support the drug study and CDx; three, clarification as to how samples and data would be managed if a patient withdraws consent at any time during or after the trial; and four, a statement that provides the pharmaceutical and diagnostic companies with the use of samples and data for the development of any future therapeutics and assays. Any confusion with the third element can lead to challenges during analysis. If possible, the fourth element should be worded such that the samples and data collected are not restricted only to the current

COMPANION AND COMPLEMENTARY DIAGNOSTICS

6.4 CLINICAL STUDY CONDUCT

131

clinical study. Both the pharmaceutical and diagnostic protocols and the ICF require review and approval from an IRB or IEC and local regulatory bodies, as applicable. Once expectations are set, a clinical project timeline should be developed between pharmaceutical and diagnostic partners, which includes time for IRB/IEC approval which may vary depending on the countries involved in the trial. As the pharmaceutical and diagnostic partners prepare for a prospective study, the joint clinical operations team should work with the CRO and investigational testing sites to create a laboratory manual that would be issued to the clinical investigators. The clinical investigators are managed by pharma and selected based on criteria such as their experience in conducting clinical studies according to the ICH Good Clinical Practice Guideline (see above) and their access to the applicable patient population. The laboratory manual may include logistical information, such as query resolution process, schedule of procedures, and turnaround time expectations. Most importantly to the diagnostic partner, the manual should include instructions on how to collect and store samples from patients as well as sample volume/size requirements and temperature criteria for shipping samples to the investigational testing site that align with the diagnostic study protocol. The investigational testing sites are managed by the diagnostic partner and selected based on criteria such as their experience with the diagnostic test, experience in conducting clinical studies according to Good Clinical Practices and Good Laboratory Practices as stated by the World Health Organization (http://www.who.int/tdr/publications/documents/glp-handbook.pdf) and the US FDA Code of Federal Regulations US FDA 21CFR part 58 (https://www.accessdata. fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart 5 58). As a check and balance, the diagnostic partner should also work with the investigational testing sites to ensure that there are internal procedures to confirm the following factors before initiating testing. The diagnostic partner needs to ensure that the laboratory’s laboratory information system (LIS) or laboratory information management system (LIMS) can capture and document sample identifiers, sample characteristics, and study results appropriately. The cost of customizing and maintaining the site’s LIS or LIMS for the study should be included in budgeting. For a retrospective study the diagnostic partner should be provided with the laboratory manual that was used by the CRO or pharmaceutical partner for sample collection. The manual should be used by the diagnostic partner to verify that the samples collected are appropriate for testing with the investigational assay. Before any study activities are performed for a prospective study, the pharmaceutical study sponsor may have an investigator meeting to train the clinical investigators on the study protocol and procedures. It is beneficial to include the diagnostic partner in this meeting in order to review sample and testing requirements, assay procedures, and the development process of an IVD test. It should be clarified to the investigators that the pharmaceutical and diagnostic companies have entered into a partnership, and the success of the program is tied to both the drug and diagnostic test performance. Drug approval would require approval of a CDx. Approval of the CDx test would require clinical utility data collected in the drug trial. During the execution of a prospective study, clinical investigators should be encouraged to adhere to the requirements of the diagnostic test and study protocol. Deviations from the drug or diagnostic protocol decrease the ability to demonstrate the safety and efficacy of the products and chances of a successful registration. An adverse event may arise if a sample that does not meet the criteria of the diagnostic protocol is used for testing and the

COMPANION AND COMPLEMENTARY DIAGNOSTICS

132

6. POLYMERASE CHAIN REACTION

result leads to randomization by the pharma partner into the drug study. Investigators should be reminded of the importance of providing the highest quality tissue samples or sufficient volume of plasma samples for testing in order to maximize the possibility of correctly identifying patient mutation status and enrolling the patient into the drug trial. The analysis phase of a CDx study is jointly managed by the pharmaceutical and diagnostic biometrics teams. The pharmaceutical database contains information such as patient demographics and clinical outcome. The diagnostic database contains sample characteristics and patient sample test results. As both teams perform quality checks and verification of their respective data sets, there invariably will be discrepancies between the two databases. The discrepancies are usually due to errors in patient-to-sample identifiers that can be resolved with the CRO and investigational testing sites. Discrepancies may also be observed between the pharmaceutical and diagnostic databases if a patient did not meet other inclusion/exclusion criteria. The pharmaceutical database would indicate a patient as a screen fail while the diagnostic database may have a valid test result. The joint biometrics teams would have to work together to ensure that their databases and analyses are aligned. Multiple meetings may be needed between the programmers and biostatisticians from both teams. The highest level of coordination between the pharmaceutical and diagnostic partners is usually seen during the postanalysis phase. Both clinical teams work on their respective clinical study reports and align their conclusions about the safety and effectiveness of the CDx. The pharmaceutical and diagnostic regulatory teams prepare their respective submissions to the regulatory agencies such as CDER, as applicable, and CDRH (for the US FDA) and time their submissions with these regulatory agencies. As the submissions are under review, the FDA may conduct inspections at the investigator and investigational testing sites to ensure that the respective clinical studies were properly conducted and managed. The joint clinical operations team should work together to address any questions during the inspections. In addition the pharmaceutical and diagnostic partners should collaborate when addressing questions that may arise from the FDA during their review. As they await coapproval, the regulatory and business teams from the pharmaceutical and diagnostic companies should work together on labeling for their respective products and plan joint commercialization activities which require the support from the global, regional, and local medical affairs teams.

6.5 SUMMARY/CONCLUSION PCR was initially envisioned as a powerful research tool for an academically oriented laboratory. As described above, refinements to the PCR technique has subsequently made it a powerful and reliable clinical laboratory technique for a variety of diagnostic disciplines such as infectious disease and genomics. With the advent of new powerful drugs which targetspecific driver mutations in the field of oncology, PCR has become a crucial partner in identifying which patients are most likely to benefit from these agents. It is equally important that the PCR test has demonstrated analytical accuracy and clinical utility in well designed and conducted clinical trials. The pathway to an approved test as an IVD or CDx by a regulatory agency is outlined above. While complex, this pathway ensures that when a PCR-based test is used in select patients for targeted therapy, the assay has demonstrated proof that it provides the data necessary for well-informed treatment decisions. COMPANION AND COMPLEMENTARY DIAGNOSTICS

REFERENCES

133

References [1] Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 1987;155:335
50. [2] Saiki RK, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988;239(4839):487
91. [3] Mullis KB. The unusual origin of the polymerase chain reaction. Sci Am 1990;262(4):56
61 64-5. [4] Mullis KB. Target amplification for DNA analysis by the polymerase chain reaction. Ann Biol Clin (Paris) 1990;48(8):579
82. [5] Saiki RK, et al. Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature 1986;324(6093):163
6. [6] Tong Y, et al. Application of digital PCR in detecting human diseases associated gene mutation. Cell Physiol Biochem 2017;43(4):1718
30. [7] Stewart CM, Tsui DWY. Circulating cell-free DNA for non-invasive cancer management. Cancer Genet 2018;228
229:169
79. [8] Crowley E, et al. Liquid biopsy: monitoring cancer-genetics in the blood. Nat Rev Clin Oncol 2013;10 (8):472
84. [9] Sacher AG, et al. Prospective validation of rapid plasma genotyping for the detection of EGFR and KRAS mutations in advanced lung cancer. JAMA Oncol 2016;2(8):1014
22. [10] Garrison Jr LP, et al. The lifetime economic burden of inaccurate HER2 testing: estimating the costs of falsepositive and false-negative HER2 test results in US patients with early-stage breast cancer. Value Health 2015;18(4):541
6. [11] Vyberg M, et al. Immunohistochemical expression of HER2 in breast cancer: socioeconomic impact of inaccurate tests. BMC Health Serv Res 2015;15:352. [12] Cheng MM, et al. The clinical and economic impact of inaccurate EGFR mutation tests in the treatment of metastatic non-small cell lung cancer. J Pers Med 2017;7(3). Available from: https://doi.org/10.3390/jpm7030005. [13] Ledford H. Cutting-edge cancer drug hobbled by diagnostic test confusion. Nature 2018;556(7700):161
2. [14] Chapman PB, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 2011;364(26):2507
16. [15] Karapetis CS, et al. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med 2008;359(17):1757
65. [16] Rosell R, et al. Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial. Lancet Oncol 2012;13(3):239
46. [17] Goss G, et al. Osimertinib for pretreated EGFR Thr790Met-positive advanced non-small-cell lung cancer (AURA2): a multicentre, open-label, single-arm, phase 2 study. Lancet Oncol 2016;17(12):1643
52. [18] Kalemkerian GP, et al. Molecular testing guideline for the selection of patients with lung cancer for treatment with targeted tyrosine kinase inhibitors: American Society of Clinical Oncology Endorsement of the College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology clinical practice guideline update. J Clin Oncol 2018;36(9):911
19. [19] Sepulveda AR, et al. Molecular biomarkers for the evaluation of colorectal cancer: guideline from the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and the American Society of Clinical Oncology. J Clin Oncol 2017;35(13):1453
86. [20] Wu YL, et al. First-line erlotinib versus gemcitabine/cisplatin in patients with advanced EGFR mutationpositive non-small-cell lung cancer: analyses from the phase III, randomized, open-label, ENSURE study. Ann Oncol 2015;26(9):1883
9. [21] Park K, et al. First-line erlotinib therapy until and beyond response evaluation criteria in solid tumors progression in Asian patients with epidermal growth factor receptor mutation-positive non-small-cell lung cancer: the ASPIRATION study. JAMA Oncol 2016;2(3):305
12. [22] Li M. Statistical consideration and challenges in bridging study of personalized medicine. J Biopharm Stat 2015;25(3):397
407. [23] Wilson EB. Probable inference, the law of succession, and statistical inference. JASA. 1927;22:209
12.

COMPANION AND COMPLEMENTARY DIAGNOSTICS