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36 | AUGUST 2014 | GENengnews.com | Genetic Engineering & Biotechnology News NGS Continued from page 34 TRANSLATIONAL MEDICINE forms during followup and relapse. Universal Oncology Test System "As Dr. Luthra demonstrated in her study, MiSeq is highly applicable for molecular testing of oncogenes," says Jennifer Stone, Ph.D., senior market manager for oncology, Illumina. The company has demonstrated conformity with the In Vitro Diagnostic Di- rective, and it has applied the CE mark to the MiSeqDx™, making it the frst and so far only sequencer cleared in Europe and by the FDA for clinical diagnostic applications. MiSeqDx will play an important role in the partnership forming between Illumina and the pharmaceutical industry. The goal of the partnership is to develop a universal oncology test system. "The current paradigm, [which emphasiz- es] single-analyte companion diagnostics, is changing," continues Dr. Stone. "Tumor ge- netics is highly heterogeneous, and it changes over time with disease progression. The abil- ity to monitor multiple analytes simultane- ously and continuously would enable us to manage cancer as a chronic disease." Illumina has already established itself as a leader in supplying NGS-based oncology tests for clinical research, with anywhere between 26 and 94 genes in the panels. A universal oncology test system would incor- porate 60–70 actionable cancer genes of spe- cifc interest to the pharmaceutical partners. Many analytes could represent targets of new drugs in the pharmaceutical pipeline. Illu- mina envisions that such a universal platform would be able to aggregate and systematize results from both academic and drug develop- ment efforts. Wide distribution of a universal test would enable broad patient access. Dr. Stone emphasizes that NGS seems to be a nearly ideal solution for oncology diagnostics because of its ability to profle multiple genes using only a small amount of starting material and to generate results within a short period of time. Continuously collected data would help to monitor the tu- mor genetic landscape and adjust therapies as needed to keep tumors from evolving. "The inherent genetic complexity of can- cer and the diversity of the pharmaceutical pipeline make it a challenge to pair drugs with patients who may beneft from them," observes Dr. Stone. "Our multiplexed tool would enable better pairing at both clinical trial and treatment stages." Illumina plans to deliver a fully regula- tory-compliant solution that will include a universal test; the MiSeqDx; and sample ex- traction, library prep, and other supporting technologies. Actionable Physician Reports "Our goal is to develop actionable se- quencing reports for physicians," says Rong Chen, Ph.D., assistant professor and director of clinical genome sequencing, Icahn School of Medicine at Mount Sinai. "These reports indicate which drugs would most likely ben- eft a particular patient, which drugs would have toxicities, and which clinical trials might enroll the patient." Dr. Chen's team builds a decision tree for each FDA-approved treatment as well as drugs that have not yet been approved but appear to have potential benefts, as indi- cated by molecular network information or in vitro or in vivo studies. A sophisticated Hadoop-based search engine combs though vast amounts of publicly available knowl- edge and integrates information at the DNA, RNA, and protein levels. "While the bioinformatics workfow to generate driver mutation analysis is very involved, the resulting report should be tai- lored to the physician's workfow," contin- ues Dr. Chen. "We received a lot of feedback from oncologists on how to provide the most useful information and layouts. In the future, we hope to automate the process to reach a critical mass of physicians and then start re- ceiving their feedback regarding success of the suggested treatment." Dr. Chen recognizes that genome inter- pretation may be confounded by inaccura- cies that arise from the sequencing process itself or related procedures such as align- ment, variant calling, and functional an- notation. Dr. Chen's team deploys multiple strategies to address the known causes of er- ror. These include sequencing on two differ- ent platforms and utilizing multiple variant callers and sequence aligners. In addition, the team manually reviews the most impor- tant calls and checks raw reads of known driver mutations to avoid false-negative calls. "Mistakes are unacceptable," insists Dr. Chen. "Treatment outcomes depend on accurate information." Dr. Chen's group continues to develop methods to integrate and translate various molecular measurements in the public re- positories into biomarkers for the diagnosis of disease. For example, the group's ActiVar database contains 110 million distinct vari- ants; 600,000 genome, exome, and genotyp- ing data points; and manually curated hu- man genetics papers. Dr. Chen's latest initiative, the Resiliance Project, aims to fnd secondary mutations that "balance" the disease-causing muta- tions. It aims to fnd individuals with cata- strophic genetic mutations that somehow remain healthy, presumably via protections afforded by yet undiscovered genetic or envi- ronmental factors. The Resiliance Project is the frst system- atic attempt to recruit "unexpected heroes" —people willing to donate their DNA in or- der to further research into mechanisms of disease prevention. "This project takes full advantage of all our previous experience in generating the highest quality sequencing and annotation workfow," concludes Dr. Chen. Overlooked Oncogenes "Next-generation sequencing has a bright future," comments Artur Kowalik, Ph.D., head of molecular diagnostics at Holycross Cancer Center in Kielce, Poland. He envi- sions a time when every newborn is geneti- cally screened for disease predisposition. According to Dr. Kowalik, enabling tech- nologies that could make mass screening cheaper and faster are already in develop- ment—which is not to stay that valuable screening applications don't already exist. Even currently available benchtop sequenc- ers can make a difference in patients' lives. The Holycross Cancer Center has a mid- sized laboratory that lacks the computational power of large genome centers. "Data analy- sis and clinical interpretation were our ma- jor roadblocks for NGS implementation," continues Dr. Kowalik. "We had to choose a platform that generated just enough ac- tionable data using commercial software." A single benchtop sequencer, a panel of 50 genes, and a standardized validated work- fow enabled rapid implementation of the NGS technology. Holycross uses NGS extensively to strat- ify patients for targeted therapy. Thyroid cancer is a common endocrine malignancy frequently associated with the oncogenic mutation in the BRAF gene (V600E). "When a patient presents with a suspicious diagnosis for malignancy from fne needle as- piration biopsy of thyroid gland, our endocri- nologists routinely call for evaluation of the BRAF (V600E) gene by Sanger sequencing/ qPCR," remarks Dr. Kowalik. In one case, Dr. Kowalik recalls, Sanger sequencing did not reveal mutations in the BRAF. Instead, the Holycross NGS panel was used to uncover on- cogenic mutations in a different gene (NRAS). This gene has a positive predictive value for malignancy of about 80%. Dr. Kowalik gave other examples when NGS assisted in fnding mutations that may be overlooked by "gold standard" technolo- gies. In Poland, 70–90% of inherited familial breast cancer results from only three founder mutations in BRCA1/2 genes. Prior to NGS introduction, the diagnostics was based on screening for these mutations only. Now Holycross has an ability to test the whole exome of BRCA1/2 genes. While the signifcance of all detected vari- ants may not be yet known, they may still guide the placement of tested individuals into preventive programs. As the analyti- cal capabilities of NGS grow and cost goes down, other family members may be tested for cancer predisposition. "Diagnoses made on the basis of whole-genome sequencing will relieve the uncertainty about breast can- cer risk," concludes Dr. Kowalik. To develop a system capable of generating actionable sequencing reports for oncologists, researchers at the Mt. Sinai School of Medicine are leveraging predictive network models that can comb through vast amounts of publicly available knowledge and integrate information at the DNA, RNA, and protein levels. The researchers also build a decision tree for each FDA- approved treatment as well as drugs that have not yet been approved but appear to have potential benets. In a case of thyroid cancer that was evaluated at the Holycross Cancer Center in Poland, Sanger sequencing did not reveal mutations in the BRAF (V600E) gene. Instead, an NGS panel was used to uncover oncogenic mutations in a dierent gene (NRAS). This gene has a positive predictive value for malignancy of about 80%.