March 15, 2015 (Vol. 35, No. 6)

Lisa Heiden Ph.D. Director of Business Development MyBioSource

Oncology Establishment Stakeholders Are Converging On New Treatment Paradigms

Oncology has risen to the forefront of genomic profiling, which is being used to identify actionable driver mutations and other markers. These markers can help clinicians design therapies and monitor patient responses, and they may be revealed by means of next-generation sequencing (NGS), a catch-all term describing massive parallel sequencing technologies generating gigabases of data.

NGS is already established as a research platform, and it is rapidly gaining acceptance as a clinical platform—at least as far as technical and scientific matters are concerned. Administratively and practically, however, NGS still faces barriers to adoption as a clinical tool. These include regulation, incorporation in clinical guidelines (NCCN, ASCO, etc.), reimbursement, and physician and patient education.

Accordingly, if NGS is to be fully embraced by the clinic, and if the potential for personalized oncology is to be realized, interested parties will have to cooperate and build a sense of shared commitment—at least, that was the sentiment expressed by several of the presenters at the Personalized Medicine World Conference, which was held recently in Mountain View, CA.

One of these presenters was John Leite, Ph.D., vice president of oncology market development and product management, Illumina. Dr. Leite emphasized the need to work with “all of the stakeholders involved in the oncology community to achieve consensus on what products can best serve this market, and to develop the evidence needed to support the clinical adoption of those products and physician/patient demand.” These stakeholders include regulators, advocacy groups, payers, and scientists.”

“To many stakeholders, adoption of NGS solutions requires clinical data that demonstrates the value of such solutions,” added Dr. Leite. “Clinical trials are the best mechanism to generate the technical and clinical data that measures change to patient outcome as a result of the implementation.”


Liquid Biopsies

“A growing tumor continuously sheds dead cells, spewing its genomic material, which goes into the blood and then the urine,”said Mark Erlander, Ph.D., CSO at Trovagene. “We use urine as a truly noninvasive liquid biopsy method for detection and monitoring of cancer patients with our Precision Cancer Monitoring (PCM) platform.

“The DNA becomes highly degraded. It’s about 145–150 bp medium length in plasma and 75–100 bp in urine. You have a degraded DNA sample, but it doesn’t really matter if the DNA is fragmented because as long as that sequence is there, you can interrogate it with NGS. Our mutant allele enrichment technology suppresses wild-type amplification, resulting in mutant sequence amplification allowing you to go from predominately wild-type to all mutant sequences.”

Driver mutations predict whether a patient will respond to a particular therapy. “We focus on stage 4 disease,” Dr. Erlander noted, “because this is where molecular-targeted drugs are first tested in patient populations, and this is also where things are most dire for the patient.” Mutation monitoring determines how a patient is actually responding. 

In a prospective blinded study, PCM detected BRAFV600E mutations from patients with histiocytic disorders, including measuring responses to BRAF inhibitors and emergence of therapy-resistant mutations. The potential of the liquid biopsy is underscored when one considers that detecting mutations in histiocytic tissue biopsies is difficult due to low tumor content and high stromal contamination.

Circulating tumor DNA (ctDNA) is attracting intense interest, Dr. Erlander observed, for two key reasons. First, ctDNA may be assessed via noninvasive tests. Second, cancer is heterogeneous. “There are multiple proliferating clones,” he insisted. “There is always a question—which biopsy do you pick? When a patient has a spreading cancer, blood and urine offer an entire picture.”

Dr. Leite concurs: “Recently, we have seen Illumina solutions being applied to new areas of oncology that have tremendous promise to patient care, and one exciting area is ctDNA analysis. ctDNA, sequenced at high depth of coverage, may allow healthcare providers to select therapies that are specific to certain genetic lesions (such as ALK translocations or EGFR mutations) even when a tumor biopsy is not available or possible. ctDNA may also allow monitoring of residual disease and of emerging, new therapy-resistant mutations.”

Mike Nolan, vice president and general manager of oncology business at Thermo Fisher, discussed his company’s distribution of Cynvenio Biosystems’ rare cell enrichment LiquidBiopsy Platform. He asserted that it complements Thermo Fisher’s Ion Torrent Personal Genomic Machine (PGM) workflow, providing customers a sample-to-genomic data solution for analyzing cell-free DNA and circulating tumor cells from a single blood draw.


Trovagene’s Next Generation SamplingSM technology can be used to interrogate an ultra-short DNA sequence (~30 bp), yielding data that can show how mutations react to therapy over time. As tumor cells die through treatment or natural processes, small fragments of DNA are released into the blood and the urine. Measuring this circulating tumor DNA offers a new way to track cancer-related gene mutations at the molecular level.

Immune Repertoire Sequencing

“Our ImmunoSEQ NGS platform is broadly applicable across all T- and B-cell cancers, irrespective of what oncogenic mutations they contain,” said Tom Willis, Ph.D., senior vice president and general manager, hematology products, Adaptive Biotechnologies. He added that the platform is capable of broad coverage because it looks at the immune cell receptor of the cancer cell, not the cancer mutation itself.

“We rely on the fact that immune cells, including cancerous immune cells, contain distinctive DNA signatures.  These are the so-called immune cell receptor sequences that are essentially unique to each normal T or B cell as well as to each T- or B-cell cancer,” Dr. Willis continued. “The tumor is composed of a clonal expansion of a T or B cell, and it is highly likely that when you sequence that material, it is going to show a very high frequency clone that is associated with that patient’s cancer.”

The ClonoSIGHT process is a laboratory-developed test for monitoring minimal residual disease (MRD). A pretreatment sample, typically a pathology lab specimen, is used to identify the sequence of the very high frequency clone, and then that sequence data is used to track the low-frequency clone after treatment for MRD detection in blood or marrow.

“One of the nice things about this approach is the uniqueness of these sequences,” noted Dr. Willis. “They are about 100 bp long in the assay, so even if new mutations emerge, as often happens, one can quite reliably affiliate that new sequence as being related to the original cancer line and track it.”

ClonoSIGHT is used by major cancers centers and pharmaceutical companies in clinical trials. “It is clearly more sensitive than flow cytometry, and it can be used in many more kinds of specimens,” added Dr. Willis.

Lanny Kirsch, M.D., senior vice president of translational medicine at Adaptive Biotechnologies, pointed to a pioneering study where ImmunoSEQ tracked the T-cell receptor status of tumor-infiltrating lymphocytes in melanoma patients treated with a programmed death-1 receptor-targeted therapy.  “The results demonstrated proliferation of pre-existing tumor-associated clonal CD8+ T cells was linked to a positive therapeutic response,” said Dr. Kirsch.

Dr. Kirsch added that “everyone is excited” about Adaptive’s recent acquisition of Sequenta. This move, the companies announced, “is expected to expedite and expand the use of novel immunosequencing products for researchers and clinicians to diagnose, treat, and monitor patients with cancer, autoimmune disorders, and infectious diseases.”


Minimal residual disease (MRD) detection and quantification using the ClonoSIGHT process involves two steps. In the first step, the ClonoSIGHT ID test, cancer cell DNA sequences are identified in a diagnostic sample. In the second step, the ClonoSIGHT MRD test, follow-up samples are screened for the previously identified sequences in order to detect residual disease. This two-step process was developed by Sequenta, a company recently acquired by Adaptive Biotechnologies.

Big Data Management

A hallmark of NGS is the generation of Big Data, a popularized phrase referring to large, complex, and unwieldy datasets. NGS and Big Data go hand in hand, and institutional genomics computing infrastructures are becoming overwhelmed with petabyte-scale sequence datasets.

“The Clarity laboratory information management system (LIMS) is an infrastructure that helps make sure a laboratory runs efficiently and smoothly,” said Michael Ball, CEO, GenoLogics. “The sample-preparation process for NGS is very complex, and the introduction of NGS into a clinical laboratory brings key challenges including meeting regulatory requirements—CLIA, CAP, and HIPAA in the United States, and ISO internationally.”

“A LIMS helps meet these requirements by workflow enforcement, positive sample tracking, and data security,” Ball continued. “It tells you everything about a patient sample—what happened to it from the moment it entered the lab, how it was processed. A LIMS also ensures that a sample never gets mixed up with anything else in the laboratory.

“We can also automate virtually any step using our software. For example, we can program liquid-handling robots. Eliminating the human interaction allows you to reduce the number of potential errors. There are laboratories using Clarity LIMS where there is very little human intervention from when a sample enters to when it gets to the sequencing process.”

“One of our unique capabilities is to build software systems to meet both research and clinical needs,” asserted Ball. “This is critical because virtually all clinical laboratories doing NGS also have a research component, and the tracking needs are very different.” For example, research and clinical environments may emphasize flexibility and regulatory compliance, respectively.

“Part of the problem with NGS Big Data is organization, just making sure that you don’t lose anything and that you know everything that was associated with that sample—every sequence file, every sequence analysis,” continued Ball.

“Our Clarity LIMS software clearly provides that interface so you know where everything resides.

“The second piece is just really about Big Data amounts. Some of our customers are going to our cloud-based LIMS implementation because it provides a much more scalable way to increase storage capacity and is usually a cost-effective way to hold on to all that data.  In the cloud, one can also readily access an abundance of computational resources.”

Dr. Willis described Adaptive’s clinical and research Big Data strategies as follows: “The Big Data challenge on the ImmunoSEQ clinical side is handled in our software pipeline. We build complete solutions that allow patient samples to go all the way through the process of generating and processing sequence data, to producing robust clinical answers for doctors in the form of MRD reports.

“Researchers, however, need access to the raw data, and our ImmunoSEQ Analyzer is a powerful intuitive cloud-based software that enables them to access and process as well as share their raw big data in various ways.” 

Mike Pellini, M.D., president and CEO of Foundation Medicine, said that his company “leverages FoundationCore, a database containing standardized comprehensive profiles on more than 35,000 patient cases.” The Big Data profiles stem from the FoundationOne and FoundationOne Heme Comprehensive Genomic Profiling NGS tests for evaluating the molecular drivers of patients’ solid tumors and hematologic cancers.

The company’s Interactive Cancer Explorer portal, ICE2, is “designed to enable physicians to increase the efficiency of patient care utilizing Foundation Medicine’s tests and enhance the utility of comprehensive genomic profiling,” remarked Dr. Pellini. “ICE2 features PatientMatch™, which leverages FoundationCore, to help increase the actionability of the tests. Using PatientMatch, physicians can connect with other physicians treating patients with similar genomic/tumor profiles to share treatment and outcome information in a compliant manner.”


Business-Business Partnerships

“Cancer is a formidable opponent, and by working together to leverage strengths in a partner model, we can develop better solutions and expedite their delivery to the community, which includes patients, care providers, and researchers,” said Nolan. “The strength of our NGS program is evident in the hundreds of thousands of results that have been generated in clinical laboratories using our Ion Torrent technology.”

“We’ve partnered with GlaxoSmithKline and Pfizer to bring NGS oncology tests forward through the regulatory authorities and into locally delivered healthcare networks where roughly 80% of cancer patients are treated,” Nolan continued. “The first test will be developed on our Ion PGM DX platform using Ion AmpliSeq amplification technology, and it will incorporate selected content from our Oncomine Research Panel, which includes over 100 cancer genes and enables simultaneous detection of single nucleotide variants, indels, copy number variants, and gene fusions.”



























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