May 15, 2017 (Vol. 37, No. 10)

Pity the genomic scientist who specializes in biomarker development, a discipline that is still being built around next-generation sequencing (NGS). This new discipline, which could be called next-generation molecular diagnostic assay validation, is currently grappling with a range of challenges: biospecimens that are rare and of uncertain quality, analytes that are present in scant quantities, and sample preparation guidelines and data handling conventions that are still in flux.

In hopes of laying a firm foundation on these shifting sands, genomic scientists gathered at the fourth annual Genomic Sample Prep, Biomarker Assay Development and Validation, a conference that recently took place in San Francisco. This event, which was organized by Cambridge Healthtech Institute (CHI), provided an opportunity for biospecimen experts and assay developers to discuss the “major challenges and latest advances in sample preparation and the validation of NGS and other advanced diagnostics assays.”

Outstanding presentations from the CHI conference are summarized in this article. Like the conference, the main body of this article will start with observations offered by  Lin Wu, Ph.D., vice president of development, Roche Sequencing Solutions. Dr. Wu chose to emphasize best practices and approaches that could bring highly sensitive and robust assays to market. In particular, Dr. Wu cited guidelines that appeared in The Journal of Molecular Diagnostics.

On March 16, a workgroup convened by the Centers for Disease Control and Prevention published a set of guidelines on molecular diagnostics (“Principles and Recommendations for Standardizing the Use of the Next-Generation Sequencing Variant File in Clinical Settings”). Less than a week later, on March 21, the Association for Molecular Pathology and the College of American Pathologists issued an oncology-oriented joint consensus recommendation (“Guidelines for Validation of Next-Generation Sequencing-Based Oncology Panels”).

Such efforts, Dr. Wu stated, “provide recommendations for validating genomic assays analytically and clinically.” Moreover, they recognize that precious patient samples are critical for advancing genomic medicine.

The importance of patient samples was also taken up by Benoit Bouche, Pharm.D., Ph.D., managing director, Trans-Hit Biomarkers. “Hundreds of R&D projects are stuck in labs because pharma companies behind projects cannot find the biospecimens needed for analytical and clinical validation of assays,” he lamented.

The problem of limited tissue, commented Ping Qiu, Ph.D., principal scientist, Merck & Co., is especially troublesome in oncology applications, which also contend with tumor heterogeneity and sequencing artifacts. “Oncology applications of sequencing technologies are still in their infancy, especially in the immuno-oncology space,” noted Dr. Qiu, who also complained of a lack of consistency in laboratory practices. “There is currently no industry standard,” he said, “in terms of reference materials, DNA/RNA extraction, sample input amounts, or bioinformatics pipelines, etc.”

The strategies pursued by different laboratories depend not only on the markers—whether DNA, RNA, or protein—but also on the types of questions that are being asked, and the technologies that are being used. Suppose a biomarker assay identifies a clinically actionable somatic mutation in a cancer. Is the mutation in 100 cells or 100% of the cells, or 10% or 5%?

A liquid biopsy technology is being developed by UCLA scientists to detect tumor-causing mutations in saliva and other bodily fluids. The technology, called electric field-induced release and measurement (EFIRM), leverages highly specific molecular probes and an enzymatic amplification system to achieve high-sensitivity detection of circulating tumor DNA and proteins associated with cancer. The EFIRM detection process takes place on a high-throughput electrode array, allowing for the testing of multiple patients and multiple cancer markers simultaneously.

“You must pinpoint and validate,” asserted Madhuri Hegde, Ph.D., adjunct professor, Emory University, and vice president and chief scientific officer, global laboratory services, diagnostics, PerkinElmer. “This knowledge will drive the drug treatment for that patient.”

Although the challenges addressed at the CHI conference remain daunting, several presenters highlighted advances in biospecimen science, including promising results with unconventional specimens. For example, David T.W. Wong, endowed professor, associate dean of research, UCLA School of Dentistry, discussed how saliva and salivaomics could be used in the detection of oncogenic mutations in human cancers. “Biomarker validation,” insisted Wong, “is the key element in biomarker research, particularly in patient research.”

“Real” Specimens in Potential Assays

Dr. Wu verifies analytical performance of next-generation biomarker assays. “We are developing targeted biomarker panels to measure tumor-specific mutations in liquid biopsies,” she said, adding that Roche is also working on a panel to measure how many tumor mutations are in the patient’s blood overall.

Analytical validation technologies are considered promising if they excel in terms of sensitivity, specificity, and reproducibility. Although performance attributes such as these are important, they do not, by themselves, guarantee success. Dr. Wu emphasized that analytical verification doesn’t prove assays have clinical utility; the biomarkers therein may not be 100% proven for clinical use yet. Analytical studies must be done prior to moving assays to clinical validation; otherwise, resources and time could be wasted on precious patient samples and trials.

“It is very important for us to complete the analytical biomarker studies before we commercialize the biomarker assays,” confirmed Dr. Wu.

Roche uses “real” clinical specimens and follows guideline recommendations such as the ones recently published in The Journal of Molecular Diagnostics. Admittedly, collection of hard-to-find real clinical specimens can be challenging and costly. Nevertheless, it’s important, and some of the analytical verification studies for a biomarker assay should be done with the “real” clinical specimen that the assay is intended to be used with, asserted Dr. Wu.

For example, genomic DNA isolated from cell lines versus tumor tissues versus a patient’s blood will all be different. “Analytical validation can’t just be done with cell line DNA,” cautioned Dr. Wu. “It’s too clean, and it doesn’t give you the variability found in sample specimens.”

It’s challenging to determine the ground truth when using clinical specimens. “You might say your assay device can measure certain copy numbers of DNA, but you need to verify your claim with an independent method,” advised Dr. Wu. “Typically, we develop more than one method to measure the same biomarker.” The biomarker is measured not only in the assay intended for the customer, but also in independent tests on the same clinical specimens. “I don’t see that being done with too many others in the industry as a standard practice,” she concluded.

Biobank Withdrawals in Support of Liquid Biopsies

“Liquid biopsy, the evaluation of circulating tumor cells (CTCs) or circulating DNA in blood samples taken from cancer patients, is currently the hottest segment within the biomarker field,” exclaimed Dr. Bouche. “Street analysts predict sales could soon exceed $10 billion from opportunities opened up by liquid biopsy technologies.”

Pharmaceutical and in vitro diagnostic (IVD) companies that are working on the same targets are also “competing” for the same biological sample types. Samples collected from patients treated by drugs targeting the PD-1/PD-L1 pathways, now just becoming available, are particularly difficult to find. “Lack of access to biospecimens is the main bottleneck for researchers,” observed Dr. Bouche.

Trans-Hit Biomarkers asserts that it offers unparalleled and fully transparent access to biospecimens, and that it has a proprietary network of over 100 clinical sites and academic biobanks. Biospecimens are collected and curated from more than 400 hospitals all over the world, including North America, Europe, and Asia.

In contrast, biospecimen brokerage firms whose core business is to provide off-the-shelf commoditized samples likely lack the depth of expertise and academic connections to meet liquid biopsy sample requirements, cautioned Dr. Bouche.

Unprecedented demand for matched plasma and formalin-fixed, paraffin-embedded (FFPE) blocks spurred Trans-Hit BioMarkers and its U.S. partner, MT Group, to initiate and sponsor a consortium in lung cancer. The consortium aims to collect 5,000 high-quality unique patient series of specimens (matched tissues and biofluids at diagnosis and relapse) within one year. The samples will be ethically collected from different geographies, annotated with detailed clinical information, and screened for biomarkers of current interest (EGFR, KRAS, BRAF, ALK, MET, ROS, RET, and PD-L1).

“Our initiative will accelerate the development of new biomarkers that are of strategic interest for public health but are stuck in laboratories due to the scarcity of high-quality samples,” concluded Dr. Bouche.

Raw and Amplifiable DNA

Mutational load (that is, number of mutations) is a predictive biomarker of response to checkpoint inhibitors such as Merck’s pembrolizumab. The more mutations a tumor has, the more likely a response. Whole exome sequencing (WES) can help define the mutational landscape and correlate mutational load with the likelihood of responding to immunotherapy.

“There’s a lot of preanalytical work that needs to be streamlined and standardized before the mutational load assay can be validated,” cautioned Dr. Qiu. For example, several parameters that are relevant to NGS-based tests—including the amount of DNA input for WES—are in need of standardization.

The lack of standardization is complicating tests that extract nucleic acids from FFPE tissue. DNA/RNA extracted from FFPE tissue is typically highly degraded, fragmented, and cross-linked.

Dr. Qiu has evaluated many different DNA/RNA extraction kits and quantification methods. This work led him to conclude that “10 different vendors might provide 10 different DNA input recommendations, ranging from 50 ng to 2 μg.” Standardization is lacking, and quantification is inaccurate, because amounts include poor quality and unamplifiable DNA.

“We investigated several more-accurate measurement methods called ‘amplifiability of the DNA’ to qualify the amplifiable copy of DNAs instead of the ‘raw’ DNA from FFPE extractions,” explained Dr. Qiu. Input guidelines based on copies of amplifiable or usable DNA can be more standardized and controllable than raw DNA input amounts.

Bioinformatics Pipelines

The bioinformatics pipeline (a set of ordered scripts that take raw data to data product to analysis results) is used to align reads from NGS assays, do variant calling, and filter mutations. “We download the reference sequence from the National Center for Biotechnology Information (NCBI) website,” informed Dr. Hegde. “And we design our own bioinformatics pipeline by writing scripts to pull regions we want to interrogate clinically.”

The major step in pipeline validation is to check your assay sequence data, such as data from 5,000 known disease-causing genes in WES assays, against the reference sequence pulled. “There should be a one-to-one sequence match if your assay was designed properly,” asserted Dr. Hegde.

When different pipelines are used for biomarker validation, results vary. “If a researcher uses five different pipelines for the same raw data, the overlapping mutation rate is only about 50%,” Dr. Qiu pointed out. “If you’re getting results from different laboratories, you cannot compare the data unless you do a unified analysis by the same pipeline.”

Whole Exomes for High Coverage

Validation for genetic biomarkers must ensure that “you are not dropping out regions of clinical importance,” insisted Dr. Hegde. “The key aspect of validation for genetic biomarkers is to make sure the assay design itself is accurate.”

Mutations in rare diseases could be anywhere in the genome. There are 22,000 genes in the human genome and about 5,000 known disease-causing genes. WES screening allows interrogation of all 22,000 genes in one assay. “The 5,000 genes must be properly annotated, not just to clarify what is happening at the gene level, but also to know whether the genes may cause a disease—and to identify which downstream steps should be taken once the disease is identified,” emphasized Dr. Hegde.

Dr. Hegde leverages NCBI’s genome annotation and also does a tremendous amount of bioinformatics validation work to identify errors and make sure gene information is accurately and appropriately coded. The WES assay design must include full, accurate coverage of the 5,000 disease-causing genes as well as inclusion of the 17,000 other genes.

For WES assay validation, clinical laboratory improvement amendments (CLIA) require establishment of a sample type, such as whole blood or saliva for DNA extraction. Dr. Hegde says they can also opt in already extracted DNA such as DNA NA12878, from NIST’s Genome in a Bottle project, which has already been sequenced and facilitates validation.

Ultimately, the bioinformatics pipeline must be able to properly annotate sequencing data obtained from clinical samples to help identify, discover, or rule out disease-causing biomarkers. Once annotation is completed, a board-certified clinical molecular geneticist will determine whether sequence variants in the rare disease sample should be classified as benign, disease causing, or indeterminable.

EFIRM Electrifies Liquid Biopsy

“Our analytical platform is called electric field-induced release and measurement, or EFIRM,” said Wong. “We are excited to bring it to liquid biopsy. EFIRM is an emerging technology with performance advantages over current digital polymerase chain reaction (PCR) and NGS technology.”

EFIRM allows for rapid and direct detection of nucleic acids, such as circulating tumor DNA mutations, in less than one drop of saliva, blood, or urine. Other liquid biopsy technologies require 10 mL blood or 100 mL urine. The National Cancer Institute recently funded a project for advancing EFIRM-liquid biopsy (eLB) to a CLIA-certified laboratory developed test for detecting actionable EGRF mutations in patients with non-small cell lung cancer (NSCLC).

EFIRM is a hybridization reaction that may be run in less than 15 minutes. Mutated sequences in the biofluid actively hybridize to an immobilized oligonucleotide capture probe sequence. Then, a horseradish peroxidase reporter/detector probe containing a complementary sequence also hybridizes to the captured mutated sequence, which is detected through a substrate reaction. “The small volume and electric field allow a very specific reaction and reporting of the mutant sequence,” Wong explained.

The electric field pulsates rapidly, lysing exosomes present in the biofluid and releasing their molecular contents, including DNA, RNA, and protein, into the biofluid. Target tumor DNA trapped within the exosomes is freed and can quickly be captured by the capture probe sequence.

“A proof-of-concept clinical validation in two small, published pilot studies showed eLB detected actionable EGFR mutations in patients’ saliva with 95% concordance with genotyped biopsies,” asserted Wong. Comprehensive EFIRM assay validation studies will be done with plasma from 300 genotyped cases of NSCLC. A reference laboratory will do an independent evaluation on the same plasma with digital PCR. “Thus, we will have a head-to-head comparison between EFIRM, genotyped samples, and digital PCR,” concluded Wong.

Biomarker Discovery Assays

Multiplex immunoassays are powerful tools for biomarker screening and discovery, and take a high level of expertise to develop and produce consistently. Three key aspects of multiplex immunoassay development include selecting biologically relevant targets and high-quality antibody pairs, constructing calibration curves for quality control, and  optimizing reagents to reduce plex-level effects and cross-reactivity between assays.

Specifically, screening for the right antigens and antibodies from the beginning significantly affects all other downstream steps. Variability in the raw materials used for these processes must be carefully tracked to reduce any subsequent impact on the finished panel, and robust multiplex assay development should employ strategies to control intra-lot variation by sourcing raw materials from the same lot whenever possible. If there is a need to dip into a fresh batch or lot, it is important to follow up with additional validation steps.

Bio-Rad’s Bio-Plex 200 system offers researchers a multiplex assay solution that is designed to permit analysis of up to 100 biomolecules in a single sample.

The new batch/lot of raw materials should be tested for concentration and activity in order to maintain consistent final assay performance. Furthermore, the final assay should be validated as a whole to ensure uniformity between lots. For example, Bio-Rad Bio-Plex multiplex immunoassays are typically validated at the raw material and finished kit levels to ensure accurate, dependable, and reproducible performance on biological samples, according to Bio-Rad officials.

Capture and detection antibody selection is also crucial to overall assay performance. Antibodies validated for single-plex ELISAs likely won’t perform similarly in multiplex assay applications and Western blotting antibodies may not be suitable in a multiplex assay either. Thus, diligent qualification and validation of antibody pairs must be carried out on the same platform used for the final assay to ensure optimum performance.

Choosing an assay with validated, fit-for-purpose antibodies that has been carefully tested for quality will generate consistently accurate results.

Autoantibodies as Biomarkers for Cancer Immunotherapies

Novel immunotherapies, such as therapeutic vaccinations and checkpoint inhibitors, have shown great potential in the fight against cancer, but are currently only effective for a subset of patients. Furthermore, as they work by stimulating the immune system, they can also trigger immune-related adverse events (irAEs), sometimes even leading to severe autoimmune diseases.

Given these challenges, there is a need to identify biomarkers that can better predict and monitor treatment response, as well as detect the early signs and the course of irAEs. However, traditional oncological biomarkers like genetic mutations have so far failed to provide the answers, as they cannot be used to monitor the activity of the immune system. As such, novel biomarkers will be required.

Autoantibodies are established biomarkers for the diagnosis, prognosis, and patient stratification of autoimmune diseases. Given the effect of cancer immunotherapies on the immune system, as well as the fact that irAEs that have been observed when administering them, autoantibodies could also be effective biomarkers for use in immuno-oncology. However, to date, they have not been systematically analyzed in cancer patients undergoing immunotherapies.

“To fill this knowledge gap, Protagen recently announced two research collaborations, one with the U.S. National Cancer Institute (NCI) and the other with the German National Center for Tumor Diseases (NCT),” said Georg Lautscham, Ph.D., chief business officer at Protagen. “Both programs will utilize Protagen’s biomarker development engine, SeroTag®, to identify new autoantibody biomarker signatures capable of predicting therapy response and detecting irAEs in patients treated with checkpoint inhibitors, therapeutic vaccines, or combination therapies.

As Jessica Hassel, M.D., of the NCT, highlights, “At least half of patients with a metastasized melanoma see no long-term benefit when given current checkpoint inhibitors. Response rates can be increased via combination therapies, but this increases the risk of irAEs, which then occur in up to 60% of patients. To overcome these challenges, we must learn more about their immune status. Utilizing Protagen’s SeroTag platform enables this insight.”

These important new collaborations stand to generate a wealth of information that will help support the effective future development, evaluation and clinical use of cancer immunotherapies.


Lisa Heiden Ph.D. Director of Business Development MyBioSource

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