INTRODUCTION

Biopharma companies worldwide are facing multiple challenges with the evolving marketplace and business practices. Throughout the past decade, the bar for regulatory approval, reimbursement, and market adoption has constantly risen, driving a shift in the drug development paradigm from “one size fits all” to Precision Medicine. More recently, Biopharma companies have felt pressure on their drug pricing, and this trend is expected to continue. Strategically directing research and clinical efforts, making the right pipeline decisions and lowering drug costs will be critical for Biopharma companies to adjust to the new realities.

FROM BIOMARKERS TO PRECISION MEDICINE: CHALLENGES AT EACH STEP OF THE WAY

Biomarkers play a critical role in enabling Precision Medicine. In particular, genomic biomarkers have become an integral part of the new drug development paradigm, especially in oncology. Yet, progress toward true Precision Medicine is slow. Genomic biomarker assay development and validation pose multiple technical difficulties. It is not easy to generate sufficiently robust data, at every stage of development, to select the compounds with the best chance of success, especially for small and mid-size Biopharma companies with limited resources. And, once drug candidates are administered, using genomic biomarkers to get an objective measurement of drug efficacy and adjust the dose when necessary remains challenging.

SYSTEMS BIOLOGY CAN UNLOCK BIOMARKER POTENTIAL

For biomarkers to truly make Precision Medicine a reality, biomarker data must enable drug makers to make the right strategic pipeline decisions at every phase. In order to develop tests based on genetic data, it is imperative to understand gene function not only on the genetic level (DNA), but also on transcriptional (RNA) and posttranscriptional (protein, Post Translational Modification) levels. Gene expression at RNA and protein levels are often correlated, however, this correlation can be far from linear, depending not only o the gene candidate, but also on external stimuli. Although initial mRNA levels can be predictive of the presence, absence, or abundance of proteins, many factors such as translation, modification, transport, regulation, complex formation, or degradation can change the dynamics of that correlation. Complementary profiling of protein and mRNA levels with respect to given perturbations, followed by analysis of the discrepancies between protein and mRNA levels for each gene, have shown that plotting these discrepancies results in data points that are clustered with respect to gene function network.1,2

It is clear that large-scale correlations of protein and genomic biomarker data can lead to a more robust systems pharmacology and Precision Medicine strategy. The first step toward that goal is integrating broad RNA biomarker screening into drug development strategies. Compared to analyzing protein function directly, which can be timeconsuming and labor-intensive, numerous mRNAs can be screened in less than a day, and the data can be used to predict protein function. For a Biopharma company, such genomic biomarker screens, in combination with targeted protein function analysis, are instrumental for understanding drug effects, devising specific treatments, and identifying therapeutic targets.

CROSS-PLATFORM VALIDATION: INTEGRATING MULTIPLE TYPES OF BIOMARKER DATA FOR BETTER DECISIONS

At EMD Millipore, a cross-validation proof-of-concept study was performed by measuring changes in mRNA and protein biomarkers using multiple detection methods, following application of the tyrosine kinase inhibitor (TKI), gefitinib, to non-small cell lung carcinoma (NSCLC) cell lines harboring various mutations in the epidermal growth factor receptor (EGFR) gene. Mutations in the EGFR gene have been associated with overexpression and hyperactivation of the receptor, which may lead to a number of cancers, including NSCLCs.3 Targeted inhibition of EGFR using small molecule drugs or antibodies, in combination with identification of genetic mutations in the EGFR pathway, is an effective approach to precision cancer therapy. We have however, only begun to uncover the vast potential that this field has to offer.

NSCLC cell lines harboring various EGFR mutations were used to determine EGFR expression and activation as well as apoptotic gene expression following gefitinib treatment. Cross-platform validation was carried out to correlate the levels of protein biomarkers as measured by flow cytometric analysis and MILLIPLEX® MAP bead-based immunoassays using the Luminex® technology.

NSCLC cell lines were evaluated for their EGFR mutational status using the QIAGEN Pyromark® Q24 Pyrosequencer. The Pyromark Q24 sequencer allows real-time detection of single nucleotide polymorphisms (SNPs), gene deletions, methylation, and microbial identifiers using the pyrosequencin technology. It is ideal for the detection of short sequences and can run 24 samples in 15 minutes. Mutation analysis confirmed that the NSCLC cell line A549 was wild-type for EGFR, the H1650 cell line had an exon 19 deletion, and the H1975 line had L858R and T790M substitution mutations. Exon 19 deletions and the L858R mutation constitute the most common mutations associated with EGFR TKI sensitivity and are also associated with a greater than 70% response rates in patients treated with the TKIs gefitinib or erlotinib. T790M substitution and exon 20 insertions, on the other hand, are responsible for acquired resistance to TKIs.4 EGFR mutant cells H1975 expressed two-fold higher EGFR mRNA as compared to wild-type.

In order to correlate the mutational status to TKIs sensitivity, NSCLC cells were treated with gefitinib and assessed for the mRNA an protein expression of apoptotic markers by qPCR, flow cytometry, and MILLIPLEX® MAP bead-based immunoassays. The qPCR analysis was conducted using a Taqman® low density array of apoptotic markers and analyzed on the Applied Biosystems® (ABI) ViiA7 real-time PCR system. Integrated with the ABI® Twister II® Robot, it is a highthroughput and automated platform for running and analyzing thousands of qPCR reactions. It is compatible with both the SYBR® Green and Taqman chemistries. Gefitinib induced higher expression of several apoptotic markers in EGFR-mutant cells as compared to wild type.

Flow cytometric analysis indicated higher total and phosphorylated EGFR in mutant cells and analysis using MILLIPLEX® MAP assays on the Luminex 200TM instrument validated the results. Apoptotic markers were also correlated on several mRNA and protein levels: Bcl-2, a prosurvival marker, was constitutively expressed at higher levels i mutant cells, however, there was no significant change in total or phosphorylated Bcl-2 following treatment with gefitinib. Collectively, the results not only provided a comprehensive overview of EGFR expression and TKI susceptibility in mutant cell lines, but they were also indicative of the regulation patterns driving conversion of mRNA to protein for the different markers. High throughput mRNA analysis enabled the screening and selection of multiple apoptotic markers for protein level analysis using flow cytometry or the Luminex platform.

ELUCIDATING MECHANISMS OF DRUG RESISTANCE: THE NEXT STEP

This EGFR study provided an excellent model for how Precision Medicine has become a reality in NSCLC therapy. A genomic biomarker-driven approach not only determined the predictive value of EGFR mutations for administration of firstgeneration TKIs, but also provided evidence of the development of resistance due to secondary mutations and therefore the need for second-generation TKIs.

Resistance to TKIs eventually develops in 30% of the treated patients and is attributable to several factors, includin secondary EGFR mutations or alterations in PI3K/Akt, IGF1R, pTEN or NF?B signaling, or increased IGF1R signaling. Other mechanisms of resistance include Met amplification, PI3KCA mutation, Axl amplification, or epithelial-to-mesenchymal transition (EMT). The T790M resistanc mutation, often referred to as the “gatekeeper mutation,” is found in 50% of the patients that develop resistance to EGFR TKIs. T790M may be conferring resistance by altered binding of TKI in the EGFR ATP pocket.5 Mechanisms to overcome resistance to EGF TKIs include either second-generation EGFR inhibitors, such as PF00299804 (Pfizer) to induce response in patients with an EGFR exon 20 insertion, or WZ4002, to specifically target T790M EGFR and inducing growth inhibition both in vitro and in vivo.6,7 Additional therapeutic strategies include using PI3K, Akt, or IKK inhibitors or an IGF1R antibody in combination with EGFR TKIs.

Extending our initial cross-platform validation study, we investigated underlyin mechanisms of resistance to erlotinib and gefitinib in NSCLC cell lines with activating or resistance-conferring mutations. Moreover, we evaluated effectiveness of PI3-AKT or IGF1R inhibitors as therapeutic approaches t antagonize resistance. NSCLC cells harboring exon 19 deletions or T790M mutation were treated with erlotinib or gefitinib and evaluated for EGFR, PI3-AKT, or IGF1 activation. Cells were treated with an IGF1R inhibitor from EMD Millipore or a PI3-AKT inhibitor and evaluated for EFGR and downstream pathway activation and susceptibility to TKIs. These studies were executed on multiple platforms to investigate expression at transcriptional and translational levels. H1975 cells with the T790M resistance mutation demonstrated more resistance to Gefitinib when compared to the H1650 cells; mRNA profile of H1975 cells showed high expression of the PIK3CA, PIK3R2 and prosurvival BCl2 whereas the proapoptotic BCl2L11, Caspase 6, Caspase 8 and Fas were expressed at very low levels. Moreover, H1975 cells expressed higher p-EGFR, p- BCl2 but low p-Akt, however, EGFR activation was inhibited following treatment with WZ4002. The cross platform analysis demonstrated correlation in expression at mRNA and protein levels, however, some markers did not correlate indicating posttranslational regulation.

EMD MILLIPORE GENOMIC BIOMARKER SERVICES: UNLOCKING THE POTENTIAL OF BIOMARKERS

EMD Millipore’s goal is to help Biopharma companies unlock the potential of biomarkers in drug development and monitoring. Building on its experience and leadership in protein-based biomarker services, EMD Millipore has added genomic capabilities to its arsenal, providing customtailored cross-platform analyses of sample or systems on the transcriptional and translational levels, as illustrated in the EGFR studies described.

In its St. Charles, MO laboratory, EMD Millipore works with Biopharma companies on exploratory studies, such as exploring genomic sequence variations, which potentially impact compound efficacy or toxicity, and evaluating differences in RNA expression to monitor drug response or toxicity. Moving into the clinic, EMD Millipore completes assay optimization and validation in compliance with GLP or CLIA requirements. At each stage of development, EMD Millipore offers Biopharma clients the opportunity to validate their data across multiple platforms, such as flow cytometry and Luminex technology, thereby enabling clients to make robust pipeline decisions.

TO DEVELOP COMPANION DIAGNOSTICS, INTEGRATE BIOMARKERS FROM THE START

As Biopharma companies successfully advance their compounds in the early phases of clinical development, their goal is to narrow down the number of meaningful biomarkers that might ultimately become companion diagnostics. Co-developing drugs and diagnostics is a landmark of achieving Precision Medicine. The US Food and Drug Administration (FDA) has recently published guidelines to that effect, recognizing the potential of such combination to address some of the challenges facing the industry.

Yet the hurdles to developing companion diagnostics are even higher than those facing biomarker development, particularly for small- and mid-size companies. Indeed, most of those companies aim at generating proofof- concept data for their compounds with limited resources and constrained timelines. By not recognizing the importance of integrating biomarkers early in the drug development process and not considering companion diagnostics as an ultimate goal, these companies are simply not ready to select a companion diagnostic partner and negotiate agreements with long-term implications.

EMD Millipore understands and addresses the need for a smooth transition from biomarkers to companion diagnostics in a timely and cost-effective manner. EMD Millipore facilitates this transition by validating assays on clinically well-established platforms, in its GLP and CLIA compliant laboratory. As a nimble CRO dedicated to quality, EMD Millipore is able to meet Biopharma companies’ tight clinical timeline, generate robust, reproducible data, and help companies reach their inflection point. EMD Millipore contributes to creating value for those companies by validating the assay that enhances the profile of their compounds. EMD Millipore strengthens their negotiating position as they seek partners to undertake the final stage of clinical development.

Genomic biomarkers will continue to significantly impact drug development and patient treatment. Biopharma companies strive to deliver on those biomarkers’ promises but face significant challenges to do so. EMD Millipore uniquely combines genomic- and proteomic-based platforms, assay development and validation expertise, quality systems, and flexibility to create value for Biopharma companies’ pipeline. In addition to the already well-established platforms, EMD Millipore keeps up with the latest technologies and developments to be able to offer the best solutions for Biopharma companies with quality and time in mind. With the development of revolutionary genomic platforms, it will not be long before physicians will be able to conduct a genetic test in the time it takes to write a prescription. Next-generation sequencers, robotic qPCR systems, and disposable microfluidic cartridges are already becoming the new normal, redefining conventional concepts of molecular biology. EMD Millipore constantly evaluates new technologies to strengthen its portfolio of biomarker services and evolves with the needs of Biopharma companies. EMD Millipore’s goal is to create value for its customers by unlocking the potential of their biomarkers and compounds.

REFERENCES

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Dr. Sirosh Bokhari’s 10 years of research experience covers molecular and cell biology, molecular genetics, molecular oncology, microbiology, and pathology. Trained at Stanford University and the University of the Punjab, Dr. Bokhari most recently served as staff scientist at Washington University in St. Louis, Missouri, before joining EMD Millipore, where she is responsible for managing the genomics biomarker laboratory operations and conducting nucleic acid testing in a GLP environment. Dr. Bokhari can be reached at (636) 720-1037 or sirosh.bokhari@emdmillipore.com.