New tools provide dramatic improvement in the treatment of cancer
By Craig C. Foreback
Ever since the completion of the Human Genome Project in 2001, human genome sequence researchers have been working to fulfill the long-awaited promise of personalized medicine. Indeed, human genome research has significantly improved the ability of clinicians to target prognoses and treatments specifically to individual patients’ needs. Next-generation sequencing (NGS) and cancer proteomics are two examples of such pioneering technologies.
Such cutting-edge applications of genomic research could have been only a dim glimmer of an idea in the mid 1970s, when Frederick Sanger and coworkers devised the earliest methods for sequencing DNA. But through general improvements in sequencing automation and other areas—such as fluorescent labeling and capillary electrophoresis—it was essentially a mass-production version of the Sanger method that was used to discover and publish the results of the Human Genome Project. It has been estimated that the cost of obtaining that first human genome sequence totaled approximately $3 billion.
Although Sanger sequencing and related technologies have been widely used, the utility of the technique has been hampered by inherent limitations to throughput, scalability, and resolution. Equally important, although processing improvements gradually reduced the cost of sequencing a single genome from $100 million in 2001 to around $1 million later in the decade, the technique has remained prohibitively expensive for everyday clinical application.
The breakthrough technology on which current advances are based—termed “next-generation sequencing”—was first introduced in 2005 and finally entered the market in 2007. It is a fundamentally different approach to sequencing, and represents a major transformation in the way that scientists extract genomic information from biological systems.
The concept behind NGS is different from the Sanger technique, in that the DNA fragments under study are identified in a massively parallel fashion. For example, in sequencing by synthesis chemistry, the bases of a small fragment of DNA are sequentially identified as each fragment is resynthesized from a DNA template strand. Rather than being limited to a single or a few DNA fragments, NGS instruments extend the process across millions of reactions.
Naomi O’Grady, senior product manager for oncology marketing at Illumina Inc, San Diego, explains that this advance enables rapid sequencing of large stretches of DNA base pairs spanning entire genomes. Currently available sequencing instruments have the capability of producing more than a terabase in a single sequencing run.
Moreover, says O’Grady, NGS is highly scalable. For sequencing small bacterial or viral genomes or targeted regions such as exomes, a user can choose a lower-output instrument such as Illumina’s FDA-cleared MiSeqDx sequencer. If the need is to process a larger number of samples, multiplexing on a high-throughput instrument such as the HiSeq 2500 or the HiSeq X Ten provides maximum throughput and lowest cost for population-scale sequencing.
Matthew Thomas, PhD, associate professor of natural science at the State College of Florida, instructs students in the 2-year biotechnology program—including NGS. “Illumina is the leading company in the development of NGS technology,” says Thomas.
THE NEW ERA OF GENETIC MEDICINE
But leading the way in genetic medicine requires more than just advanced technologies; it also means working to make them affordable and accessible to patients whose conditions may benefit from the promises of personalized medicine. Here also, Illumina has been a leader in the field, announcing in January of this year that its high-end HiSeq X Ten sequencer was capable of sequencing a complete human genome for less than $1000.1,2 With its ability to generate large volumes of data quickly and at a reasonable cost, NGS has rapidly found its way into transplantation medicine, microbiology, inherited disease, reproductive health, and oncology.
Traditionally and currently, cancer is diagnosed primarily through pathological staging and, in some cases, molecular cytogenetics. But cancer is a very complex disease of the genome that encompasses a wide variety of genetic alterations, including translocations, amplifications, and point mutations. By looking at such genetic alterations, NGS has the ability to transform the focus of cancer diagnosis and treatment from an organ basis to a molecular basis. Looking at the genetics of a cancer, NGS can identify the type of mutations present in a cancer sample, which can be used to inform therapy selection. NGS may also be able to distinguish between primary and secondary tumors, and identify the source of metastases.
Cancer genes are dynamic and can change in response to therapy. Because cancer genes are not stable, one snapshot is not enough. We know that an accumulation of mutations may lead to cancer, and that some mutations can change or even diversify the cancer. Some changes alter aggressiveness, which may make a cancer more treatable; while other changes may alter different responses, leading to relapses. NGS can be applied to understand the genetic basis of the disease. “Germline risk prediction, companion therapeutics, and molecular monitoring of circulating nucleic acids are the focus of Illumina’s cancer business,” says O’Grady.
To prepare for the regulated future of NGS, Illumina has acquired Myraqa, a regulatory and quality consulting firm specializing in in vitro diagnostics, with emphasis on companion diagnostics. “Myraqa is recognized as the leader in regulatory and quality matters in molecular diagnostics,” says Rick Klausner, MD, chief medical officer at Illumina. The acquisition is part of Illumina’s commitment to making itself into a pre-eminent clinical company.
THE PROTEOMIC AGE
“Clinical proteomics” involves the application of proteomic technologies on clinical specimens such as blood. Cancer, in particular, is a model disease for applying such technologies in order to identify unique biosignatures and biomarkers useful for the diagnosis, prognosis, and therapeutic guidance of the disease. Biomarkers are biological molecules found in tissues, blood, or other body fluids, and constitute signs representing a normal or abnormal biological process, or a condition or disease. They may also be used to see how well the body responds to treatment for a disease or condition.
Boulder, Colo-based Biodesix Inc, a fully integrated molecular diagnostics company, is leading the pack by forging the path toward the proteomic age. “Physicians will increasingly use diagnostic tools to provide dramatic improvements in the treatment of their patient’s disease,” says David Brunel, Biodesix CEO. In 2009, Biodesix released its first protein-based diagnostic test, which analyzes proteins instead of genes to better match patients with treatments.
Lung cancer is the most common cancer in the world, and the survival rate is typically low. In developing its first test, Biodesix saw a need for a quick diagnostic test that could help oncologists prescribe second-line therapy choices for patients with advanced non–small cell lung cancer (NSCLC). In this case, it was the drug Tarceva (erlotinib) versus single-agent chemotherapy.
To develop the VeriStrat test, a team of Biodesix physicists and mathematicians created a set of analytical algorithms and processes collectively called deep MALDI (matrix-assisted laser desorption/ionization). “VeriStrat represents a unique advance in the field and is one of the first tests that analyzes the phenotype instead of the genotype,” says Brunel. “VeriStrat classifies patients with different protein profiles, providing oncologists with important information for managing their lung cancer patients.” This analysis enables the routine use of MALDI mass spectrometry, so it is robust and reproducible enough for use in clinical diagnostics.
VeriStrat has been studied in more than 80 clinical trials and ordered for well over 5,000 patients. Results from a recent Phase 3 study of 285 patients, called PROSE, showed that participants were classified into either a VeriStrat-good or VeriStrat-poor group with regard to the use of erlotinib. Those in the poor group demonstrated better outcomes on chemotherapy compared to erlotinib.3
The use of VeriStrat to assist in selecting lung cancer therapies is just the first application for the test. It has also been used to predict patient outcomes associated with breast cancer and colorectal cancer as well as head and neck cancers. Biodesix is interested in building new partnerships with academic institutions, pharmaceutical companies, and biotech firms to explore and harness the proteomic frontier.
“Proteomics in general is the frontier,” says Paul Beresford, Biodesix vice president for business development and strategic marketing. “But additional indications for VeriStrat with different drugs within NSCLC—or outside of lung cancer in breast, colorectal, and head and neck cancers—are the future.”
Recent developments in oncology diagnostics are continuing to advance the field. “There are many early-stage technologies that show promise in a research setting for identifying cancers from a patient’s blood,” says Thomas J. Albert, PhD, vice president and head of sequencing research at Roche Diagnostics, Indianapolis. “New technologies that analyze circulating tumor DNA, circulating tumor cells, exosomes, proteins, and autoantibodies with increasing sensitivity and scale have the potential to identify more powerful screening markers.
“Stanford researchers recently published work showing that targeted sequencing of circulating DNA using Roche’s NimbleGen sequence capture technology coupled with sequencing was able to identify tumor-specific mutations in all stage II through stage IV NSCLCs, and half of the stage I NSCLCs that were studied,” Albert notes.4
Autoantibodies also have significant potential for early cancer screening. It is well accepted that the immune system reacts very early to cancer growth, recognizing tumor-specific antigens. “Roche has developed a novel peptide array technology that has essentially all the epitopes of the human proteome, represented as 2.9 million 12-mer peptides, on a single chip,” says Albert.5 “We are using this technology in collaboration with a major cancer center to research blood-based autoantibody profiles specific to lung cancer. This study is in an early stage, but shows promise in identifying potential early cancer biomarkers.”
In July, Roche Molecular Diagnostics and AstraZeneca announced a collaboration to develop a companion diagnostic test for use with tissue and plasma specimens, to identify NSCLC patients eligible for AstraZeneca’s third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), AZD9291, which is currently in clinical development.6
NSCLC patients who have been treated with EGFR-TKIs, and whose disease has progressed, typically have to undergo a repeat biopsy to assess whether they have developed a specific resistance mutation, T790M, explains Albert. “The Cobas EGFR mutation test for use in plasma is being used in clinical investigations to determine its potential to provide an alternative method of identifying the T790M mutation in order to aid in the selection of patients who may be eligible for treatment with AZD9291,” he says.
The partnership with AstraZeneca is the first step toward achieving Roche’s vision of supporting innovative companion diagnostics through the development of allele-specific PCR plasma-based testing. Through this collaboration, Roche is investigating the potential to improve management of patients with targeted therapies based on the molecular analysis of a less-invasive sample.
In addition to working with AstraZeneca, Roche Molecular Diagnostics is continuing to collaborate with Roche Pharmaceuticals on the development of new plasma-based biomarkers for cancer detection and therapy, and is assessing options on how to further personalize treatment decisions.7 “In principle, our companion diagnostic tests can help predict the efficacy of a targeted therapy—for instance, the Cobas BRAF mutation test with Zelboraf, and the CobasEGFR mutation test with Tarceva,” says Albert.
In another recent study by researchers at Memorial Sloan Kettering, targeted sequencing of circulating DNA from a breast cancer patient identified mutations that were specific to metastatic tumors and not identified in the primary tumor.8 The study suggests that the information found in circulating DNA may be more representative than sequencing the primary tumor alone, and may indicate the presence of metastasis. Both this study and the Stanford study mentioned earlier showed a clear relationship between the level of circulating tumor mutations and disease response as measured by imaging.
Biodesix is also pursuing partnerships to develop companion diagnostics. Aveo Oncology and Biodesix are partnering to codevelop and commercialize Aveo’s hepatocyte growth factor inhibitory antibody, ficlatuzumab, with a Biodesixcompanion Veristrat diagnostic test, for treatment of NSCLC.9
Illumina is also exploring companion diagnostics, and part of its goal is also to partner with pharmaceutical companies to that end.
According to Eric Lin, MD, PhD, Illumina’s medical affairs manager for in vitro diagnostics, NGS can be used to perform liquid biopsies, which measure circulating tumor DNA (ctDNA). NGS offers a method for cancer detection and molecular monitoring through ultrasensitive assessment of ctDNA.4
A recent study conducted by researchers at Cambridge University added to the growing body of research demonstrating the validity of liquid biopsies. Using NGS techniques, the study examined patient samples covering multiple classes of somatic alterations. The technique identified mutations in more than 95% of tumors, detected ctDNA in 100% of patients with stage II through stage IV NSCLC and in 50% of patients with stage I disease, and was shown to have 96% specificity for mutant allele fractions.10 The ctDNA levels were highly correlated with tumor volume and were able to discriminate between residual disease and treatment-related imaging changes. Measurement of ctDNA allowed for earlier assessment of cancer patients than traditional radiographic procedures.
Roche’s Albert and Biodesix’s Beresford agree that the developing field of liquid biopsies may hold significant advantages over traditional care procedures. Currently, patients may be required to undergo multiple invasive biopsies so that their condition can be monitored. Liquid biopsies may not only provide diagnostic data earlier, but may also enable patients to avoid repeat tissue biopsies.
Tumors are highly complex genomic aberrations that are further complicated by heterogeneity. But advancing technologies hold the potential to resolve such complexities.
The future of oncology diagnostics is being transformed by genomics and proteomics. The implementation of new molecular diagnostic techniques will result in earlier detection, precise diagnosis, and targeted therapies.
Craig C. Foreback, PhD, is a contributing writer and member of the CLP editorial advisory board. For further information, contact chief editor Steve Halasey via [email protected]
1. Illumina introduces the HiSeq X Ten sequencing system. San Diego: Illumina, 2014; available at: http://investor.illumina.com/phoenix.zhtml?c=121127&p=irol-newsArticle&ID=1890696&highlight=. Accessed August 13, 2014.
2. Lin E. On the forefront of transformation: genomic applications in oncology. Webcast 28 May 2014; available at: www.illumina.com/events/webinars/archive.ilmn. Accessed August 13, 2014.
3. Gregorc V, Novello S, Lazzari C, et al. Predictive value of a proteomic signature in patients with non-small-cell lung cancer treated with second-line erlotinib or chemotherapy (PROSE): a biomarker-stratified, randomized phase 3 trial. Lancet Oncology. 2014;15(7):713–721; doi: 10.1016/S1470-2045(14)70162-7.
4. Newman AM, Bratman SV, To J, et al. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nature Medicine. 2014; published online 6 April 2014; doi: 10.1038/nm.3519.
5. Forsström B, Axnäs BB, Stengele KP, et al. Proteome-wide epitope mapping of antibodies using ultra-dense peptide arrays. Mol Cell Proteomics. 2014;13(6):1585–1597; doi: 10.1074/mcp.M113.033308.
6. AstraZeneca and Roche announce partnership to develop companion diagnostic test for AZD9291. London: AstraZeneca, 2014; available at: http://www.astrazeneca.com/Media/Press-releases/Article/28072014–diagnostic-collaboration-cbrs-layout-2. Accessed August 13, 2014.
7. Koch WH. Zelboraf: the codevelopment of Zelboraf and its companion diagnostic. Presentation to Biomarkers & Diagnostics World Congress 2012 (Philadelphia: Cambridge Healthtech Institute, 22 May 2012); abstract available at: http://www.biomarkerworldcongress.com/bet. Accessed August 13, 2014.
8. De Mattos-Arruda L, Weigelt B, Cortes J, et al. Capturing intra-tumor genetic heterogeneity by de novo mutation profiling of circulating cell-free tumor DNA: a proof-of-principle. Ann Oncol. 2014;Epub ahead of print 9 July 2014; pii: mdu239.
9. AVEO and Biodesix partner to codevelop and commercialize ficlatuzumab with a companion diagnostic for treatment of NSCLC. Boulder, Colo: Biodesix, 2014; available at: http://www.biodesix.com/aveo-and-biodesix-partner-to-co-develop-and-commercialize-ficlatuzumab-with-a-companion-diagnostic-for-treatment-of-nsclc/. Accessed August 13, 2014.
10. Murtaza M, Dawson SJ, Tsui DWY, et al. Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature. 2013;497(7447):108–112; doi: 10.1038/nature12065.