Think of biomarker development as an offensive drive in football that proceeds from the first line of scrimmage and keeps going until it finally crosses the goal line. It’s a series of plays that includes biomarker discovery, analytical validation, clinical translation, evaluation, and implementation. Along the way, all the players on Team Biomarker—biopharma companies, antibody developers, imaging specialists, device manufacturers, central laboratories, etc.—contribute to the overall effort.

No two drives are quite the same because Team Biomarker plays against diverse opponents. For example, when attempting advances in precision oncology, Team Biomarker must recognize that no two tumors are identical. The playbook that worked well before may fail in new situations. Also, once-reliable players may need to be benched to make room for new talent.

If a biomarker, an indication of the presence or severity of a biological condition, can be used to predict whether a therapeutic will benefit an individual patient, it may qualify as a companion diagnostic (CDx). The first approved CDx—the HER2 assay—appeared in the late 1990s. It detects the overexpression of ERBB2 to predict which breast cancer patients are candidates to receive trastuzumab (Herceptin). Since the HER2 assay was introduced, the scope of potentially therapy-defining genetic differences has drastically increased. In a sense, biomarker development has come to incorporate sophisticated opposition analysis.

Which mutations or alleles present in a patient’s tumor might determine the success or failure of a checkpoint inhibitor or other oncologic drug? Even if provisional answers to such questions can be ventured, definitive answers—reliable CDx assays—often prove elusive. In the middle of a difficult drive, Team Biomarker might be tempted to punt, or emboldened to run on fourth down, depending on the nature of the opposition as well as the strengths and weaknesses of the team’s players.

Multiplex assays for analyzing tissue samples

“On the journey from assays to bedside, biomarkers pass through a development continuum,” says Philippe Mourere, senior vice president, commercial operations, Ultivue. This continuum, he notes, may be divided into stages such as “analytical validation, assay standardization, and demonstration of clinical utility on cohorts of retrospective samples.” Ultivue’s proprietary fluorescent labeling technology (described in the June 2018 issue of GEN) is positioned to help researchers rapidly advance biomarkers along this continuum until they stand at the threshold of the regulated environment.

Ultivue specializes in creating data-rich informative biomarker panels that transform traditional immunohistochemistry (IHC) from visual morphology to data-based analysis. Qualitative visual assessments are being replaced by quantitative digital pathology.

Mourere maintains that detection and quantification of biomarkers, as well as gaining an understanding their spatial relationships, is just the first step in analytical verification of a multiplex IHC assay. To ensure successful global deployment, the new tumor characterization assays must be easily implemented without significant additional resources or costly equipment.

“Seamless incorporation of a new technology into Clinical Laboratory Improvement Amendments (CLIA)-certified labs for the development of an assay requires the technology to be robust and reproducible,” he explains. “Removing economic barriers, such as additional capital expenditure for specialized equipment, is also critical for large-scale adoption.”

Another signpost along the development path is “biological rationale.” To establish a biomarker’s biological rationale and potential clinical utility, Ultivue collaborates with experts in disease biology. If the disease is cancer, Ultivue helps determine whether a molecular signature exists that predicts pathology or the response to therapy. For example, enhancement of T-cell activation to elicit an antitumor immune response is a goal of several new investigational drugs.

Measuring the pharmacological effects of these compounds requires functional assessment of both immune cells and cancer cells. Such a functional assessment was demonstrated in a study (J. Clin. Oncol. 2019; 37(15): 2629.) that represented a collaborative effort involving Ultivue, the Frederick National Laboratory for Cancer Research (the Clinical Pharmacodynamics Biomarker Program), and the National Cancer Institute (the Developmental Therapeutics Clinic/Early Clinical Trials Development Program and the Division of Cancer Treatment and Diagnosis).

In this study, a multiplex immunofluorescence assay based on Ultivue’s InSituPlex® technology was evaluated for its ability to measure T-cell receptor activation, intracellular signaling, and net stimulation/inhibition of CD8 cells. In addition to quantitating these factors simultaneously, the assay revealed the spatial relationship between CD8 cells and tumor tissues.

The authors of the study indicated that the assay “was clinically validated using custom tissue microarrays containing tumor biopsies of three different histologies [colorectal cancer, non-small cell lung cancer, and breast cancer].” They also noted that the assay “is being used for pharmacodynamic evaluations in ongoing immunotherapy clinical trials.”

“The detection of cell phenotypes requires more than one biomarker and more than a simple visual observation,” Mourere declares. “Ultivue works to achieve the domain leadership position in fulfilling the vision of capturing these complex granular phenotypes.”

Antibodies for recognizing fine distinctions

Abcam focuses on supporting the diagnostic and therapeutic parts of the biomarker development continuum by honing capacity to rapidly develop custom antibodies for important targets, including those in immuno-oncology. As a host for production of monoclonal antibodies, the rabbit has several advantages. The rabbit immune system optimizes antibodies by using two mechanisms of affinity maturation: gene conversion and somatic hypermutation. This means that rabbit antibodies are better at distinguishing subtle differences, such as epitope variations, post-translational modifications, and conformational changes.

non-small cell lung cancer
This image from Abcam shows the expected expression of PD-L1 on non-small cell lung cancer formalin-fixed paraffin-embedded tissue. PD-L1 is stained with recombinant rabbit monoclonal to PD-L1, clone 73-10 (ab228415), and detected using anti-rabbit polymer horseradish peroxidase with 3,3’-diaminobenzidine (brown). Hematoxylin was used as a counterstain (blue).

This is particularly important when developing antibodies that target a difference of just one amino acid, such as the new anti-PD-L1 antibody clone MKP1A07310 (clone 73-10) developed by Abcam in collaboration with Merck KGaA (Darmstadt, Germany). PD-1 and its ligands are playing an important role in regulation of the activity of different T-cell types. Blockade of PD-1–PD-L1 interaction provides an effective approach for specific tumor immunotherapy.

“We bring immunohistochemistry early into the triage process,” says Will Howat, PhD, director of antibody validation and characterization, Abcam. “For each target, we may screen 30–40 clones against a multiplicity of materials, including tissue microarrays, in a variety of applications relevant to the target of interest.

“Clones that pass this validation step can often also be used to create a recombinant version of this antibody, which in turn is validated in the relevant applications and species. We can also utilize next-generation sequencing (NGS) to mine the breadth and depth of the rabbit immune response.”

Mining NGS data increases the chances of finding a clone with specificity for a single point mutation. Lead antibodies, with defined biochemical and functional characteristics, are identified using tailor-made assay cascades.

“It can be a challenge to find optimized antibody clones that work very well in IHC applications that are also useful in diagnostics development, like the PD-L1 (73-10) clone,” Howat notes. “It really is an art form in itself.”

To enable these foundational studies by members of the scientific community, both diagnostic and academic scientists, Abcam has made its proprietary and licensed PD-L1 clones available along with other well-characterized antibodies to cancer and immuno-oncology biomarkers that may form a composite biomarker signature. “Abcam,” asserts Howat, “is built on a principle of providing the best available recombinant monoclonal antibodies, accompanied by comprehensive and open data.”

A central lab model for addressing global trends

The central laboratory plays a key role in developing, validating, and executing trials for companion diagnostics. “Execution of the test at the same level of quality in multiple laboratories spanning key geographies is essential to success,” says Patrick Hurban, PhD, senior director and global head of translational genomics, Q2 Solutions.

Hurban notes that clinical trials are becoming more globalized and that the matrix of diagnostic tests and targets is growing more complex. These trends, he asserts, present “a host of logistical and scientific challenges for our biopharma and device partners.”

Q2 Solutions Operational Complexity
Q2 Solutions, a clinical trial laboratory services organization, observes that clinical trials for companion diagnostic (CDx) tests require close coordination between the pharma, the device sponsor, and the testing laboratory. Protocol design, database and kit creation, sample collection and logistics, and results reporting have become more challenging with novel trial designs and testing modalities. Accordingly, says Q2, a laboratory partner with a global laboratory footprint and relationships with clinical sites could facilitate CDx trials.

 

Q2 Solutions has built a global laboratory footprint to meet the logistics challenge head-on, ensuring that samples can be delivered from over 180,000 clinical sites to the central laboratory with stability, often within one day. Partnering with local, established diagnostics laboratories capitalizes on local knowledge to overcome issues of geography and regulatory landscape. A recently established strategic partnership between Q2 Solutions and Guangzhou KingMed Diagnostics (China) will pave the way for biopharma companies that wish to launch companion diagnostics in China.

Q2 Solutions is meeting the scientific challenge through a Centers of Excellence model, where high-level expertise is concentrated in a single location that acts as a clearinghouse for complex diagnostic testing workflows. In close collaboration with biopharma clients and device manufacturers, the Centers of Excellence engage their specialized scientific teams to operationalize the test in anticipation of harmonizing testing across all Q2 global sites.

“The art of creating a CDx test involves a multidisciplinary team that consults with the client to develop test specifications that are aligned with regulatory requirements and market realities,” explains Hurban. “Because of our considerable expertise in this field, we are able to provide a comprehensive solution with detailed specifications for sample collection and preparation, shipping and stability, the appropriate analytical platform, and myriad other details. A cohesive plan increases the probability of success not only for the diagnostic test itself, but also for the entire clinical development strategy.”

Early engagement with clients ensures that all stakeholders are aligned around shared goals, and it allows for iterative development of a robust assay that does not rely on a finicky detection process. “Continuous innovation,” emphasizes Hurban, “is at the core of being responsive to the evolving needs of CDx development strategies.”

Single-site premarket approval of CDx tests

Lung cancer remains a leading cause of cancer deaths worldwide, with high mortality largely attributed to diagnosis occurring late in the disease process. “[Our] strategy is to answer clinical questions in lung cancer using blood-based diagnostics,” says Gary Pestano, PhD, chief development officer, Biodesix. “We identify and develop products covering the entire lung continuum of care, by using both an in-house development strategy and collaborative opportunities.”

Biodesix views late-stage non-small cell lung cancer as particularly underserved from a diagnosis and prognosis standpoint. Up to 30% of advanced non-small cell lung cancer patients are not eligible for tissue biopsy, and even if a biopsy is performed, tumor heterogeneity may result in nonactionable data. By then, patients likely have accumulated multiple mutations that may significantly affect the course of disease or diminish the effectiveness of therapy.

Biodesix Lung Reflex
The Biodesix Lung Reflex, a genomic and proteomic test, has a workflow that begins with a blood draw shipped at ambient temperature to the clinical laboratory. Test samples are accessioned and processed to isolate circulating nucleic acids, both cfDNA and cfRNA. Following nucleic acid extraction and cDNA synthesis, samples undergo library preparation and quantification using the Total Nucleic Acid (cfTNA) assay. Templating occurs overnight on the Ion Chef™ system. Sequencing is then performed on the Ion GeneStudio™ S5 Prime, and analysis is completed with a bioinformatics workflow to make variant calls.

 

A recent collaborative effort aims to bring a research test, developed by Thermo Fisher Scientific, into the clinical diagnostic space. Pestano highlights the key advantages of the Ion Torrent™ Oncomine™ Pan-Cancer Cell-Free Assay, such as easy sample collection and shipment at ambient temperatures; high concordance between NGS and reference methodologies; and consistency between runs, chips, and assays. The assay detects 52 genes across multiple tumor types, including lung cancer.

“Biodesix will provide analytical and clinical validation while obtaining FDA approval to bring this product to clinical practice,” adds Pestano.

The chosen regulatory pathway is via a single-site premarket approval. This pathway is particularly appealing in the precision medicine diagnostics space, which benefits from streamlined development and reduced upfront investment. FDA approval of an NGS test is critical for the test’s market success in light of a decision memo (CAG-00450N) issued by the Centers for Medicare and Medicaid Services (CMS). The memo specifically addresses coverage for NGS-based testing in cases of advanced late-stage cancer.

“An FDA-approved, reimbursable test performed at our specialized CLIA laboratory and supported by clinical evidence would generate a high degree of confidence among the physicians,” Pestano declares. “It may radically transform lung cancer standard of care.”

Pharma partnerships in CDx development

Rain Therapeutics offers a pharmaceutical partner perspective on biomarker development and validation. “Our focus is precision oncology,” says Vijaya Tirunagaru, PhD, the company’s vice president and head of biology and nonclinical development. “We undertake a new drug discovery and development program only if a patient population could be clearly defined by a biomarker. A well-defined CDx test could drastically improve probability of success.”

When Rain Therapeutics in-licensed tarloxotinib, an EGFR inhibitor, the company positioned it in a unique oncology niche. In-frame insertion of three or more base pairs in exon 20 of the EGFR gene is a known oncogenic driver in non-small cell lung cancer, occurring at about 9% frequency. These insertion mutations are associated with resistance to targeted EGFR inhibitors and correlate with a poor patient prognosis.

What complicates targeted product development is that EGFR with the insertions behaves pharmacologically exactly like wild-type EGFR. Compounds that inhibit wild-type EGFR cause severe toxicities.

“Our unique challenge was to develop a compound targeting the insertion mutants,” adds Tirunagaru. Tarloxotinib is a prodrug of a potent EGFR inhibitor that converts into the active drug only in low-oxygen (hypoxic) conditions observed in solid tumors. In the presence of oxygen, the inactive tarloxotinib is stabilized, avoiding the production of the active drug in healthy, oxygenated tissues.

A key CDx test identifies EGFR insertions and is used for initial patient screening. However, Rain Therapeutics is pursuing another diagnostic test that could provide additional correlation between patient genetics and tarloxotinib efficacy.

The Limits of Circulating Tumor DNA as a Biomarker

Although circulating tumor DNA (ctDNA) is a promising biomarker for monitoring treatment response in patients with various cancers, oncologists should understand its limitations. At the 2019 ASCO Annual Meeting, Jenny H.J. Lee, MD, of Macquarie University in Sydney and the Melanoma Institute Australia, and her colleagues presented a poster paper that documented the limitations of using ctDNA to understand advanced melanoma progression when it has spread exclusively to the brain (J. Clin. Oncol. 2019; 37: abstr. 9581).

The researchers used Bio-Rad’s Droplet Digital PCR (ddPCR) technology to analyze ctDNA in 48 patients with advanced melanoma and brain metastases receiving immune checkpoint therapy. Eight had metastases only in their brains. The researchers found that an absence of detectable ctDNA at the start or through the first eight weeks of treatment was a good sign, associated with superior progression-free survival and overall survival. However, this relationship did not apply to the eight patients with only brain metastases.

The researchers concluded that although ctDNA could be useful for monitoring treatment efficacy in metastatic melanoma, it may not be useful for monitoring whether brain tumors respond to immune checkpoint therapy, possibly because the blood-brain barrier filters out the brain ctDNA before it reaches the bloodstream. With up to half of melanoma patients exhibiting brain metastases at some point in treatment, this study has important implications for ctDNA as a biomarker.

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