Long-Read DNA Sequencing Uncovers Hidden Cancer Drivers Missed by Standard Tests

Two new studies from University of Michigan researchers show that long-read DNA sequencing can uncover genetic alterations that conventional sequencing misses, from hidden disruptions of tumor-suppressing genes to the molecular origin of a rare tumor that arose in a young boy after gene therapy. The findings, published in Cancer Discovery and the New England Journal of Medicine, indicate that the way mutations are physically arranged on DNA — not just their presence on a mutation list — may be a missing layer of precision oncology.

The Michigan Oncology Sequencing Program, known as MiOncoSeq, was the first program of its kind in the nation to sequence patient tumors. Launched in 2010, it analyzes DNA and RNA from both normal tissue and tumors to identify alterations that could help guide treatment decisions. In the new Cancer Discovery study, the researchers enhanced that process with long-read nanopore sequencing, a technology capable of detecting alterations that might be missed by conventional sequencing.

The distinction between the two technologies is central to the findings. Conventional short-read sequencing examines small pieces of DNA, typically a few hundred DNA letters long. Long-read sequencing, by contrast, can read thousands to tens of thousands of letters in a single stretch. That extended reach allows researchers to see complex changes in the genome and, crucially, to understand how those changes fit together — information that is often invisible when DNA is chopped into tiny fragments and reassembled computationally.

“Having a list of mutations doesn’t always explain a tumor,” said Arul Chinnaiyan, M.D., Ph.D., S.P. Hicks Professor of Pathology and Urology, director of the Michigan Center for Translational Pathology and a Howard Hughes Medical Institute investigator. “We need to understand how these mutations are arranged, which conventional sequencing often cannot resolve.”

One reason arrangement matters is that most genes come in two copies, one inherited from each parent. Nanopore sequencing can help clinicians determine whether one or both copies of a gene that protects against cancer have been disabled — something that can occur through complex DNA rearrangements and chemical modifications. It can also reveal whether multiple mutations occur together on a single copy of a cancer-promoting gene, a configuration that could potentially increase the gene’s activity or contribute to drug resistance.

In one arm of the Cancer Discovery study, the researchers screened 768 patients with prostate, breast or ovarian cancer and identified 46 tumors with signs of genomic instability whose underlying causes remained unexplained by conventional sequencing. Long-read sequencing resolved 32 of those cases by identifying hidden alterations or chemical changes affecting genes that protect against cancer. For those 32 tumors, the long-read approach identified a culprit that standard methods had not found.

In a broader analysis, the team screened 4,496 tumor samples from 3,688 patients enrolled in the MiOncoSeq program. They then applied nanopore sequencing in selected cases to determine how mutations were arranged in genes including NOTCH1, PDGFRB, KIT and PIK3CA. “Identifying these mutations can help us understand why certain patients might develop drug resistance and how we can make better treatment choices,” Chinnaiyan said.

The study also demonstrated that long-read sequencing can uncover inherited cancer risk that conventional testing misses. One woman with a strong family history of breast and ovarian cancer had previously tested negative on a commercial multigene panel. Nanopore sequencing of a blood sample revealed a disease-causing DNA insertion in BRCA2, a gene involved in repairing damaged DNA. The authors are hopeful that findings like this could help guide genetic counseling and testing for relatives when a family’s cancer history remains unexplained.

In the separate New England Journal of Medicine report, the team used nanopore sequencing to help explain how a rare tumor developed in a 3-year-old boy after gene therapy. The child had been diagnosed with Krabbe disease, a rare inherited disorder that affects the protective coating of nerve cells. As part of a clinical trial, he received gene therapy following a stem cell transplant. The therapy used a modified adeno-associated virus, or AAV, to deliver a working copy of the GALC gene, which is defective in Krabbe disease. Twenty-six months after the gene therapy, routine imaging revealed a small tumor beside his spine.

Through nanopore sequencing, the researchers found that a fragment of DNA from the modified AAV had inserted into the GLI1 gene. The inserted fragment contained genetic switches that drove unusually high activity of GLI1, which can promote tumor growth when abnormally activated. At the time of the report, the child had no tumor-related symptoms or neurological deficits and was being monitored with serial imaging. “Gene therapy holds great promise, and understanding complications like this can help guide safer design,” Chinnaiyan said. “It is also important to monitor patients over the long term after they receive treatment.”

Together, the two reports illustrate a common theme: the physical architecture of the genome can carry clinical information that fragment-based sequencing cannot capture. In the oncology setting, resolving which copy of a gene carries which mutations — a process known as haplotype phasing — explained tumors whose genomic instability had no identifiable cause under conventional sequencing. In the pediatric case, the same technology pinpointed a single viral insertion event and its functional consequence in a patient’s tumor. The Cancer Discovery work was conducted as an observational study of patient samples from the MiOncoSeq program, and the authors note that identifying these arrangements could inform understanding of drug resistance and treatment selection. Additional authors on the reports include Josh N. Vo, Yi-Mi Wu, Sophie Yeung, Mingyu Park, Yelena Kleyman-Smith, Guo Ci Teo, Alyssa J. Wu, Anne Li, Jamie C. Estill, Lakshmi P. Kunju, Chen Yang, Amer Heider, Xuhong Cao, Rui Wang, Tiffany Pham, Suresh N. Magge, Hemant Parmar, Rajen Mody and Dan R. Robinson. The research was supported in part by National Cancer Institute grants, including a Prostate SPORE grant, the Early Detection Research Network, a Research Specialist Award and an Outstanding Investigator Award to Chinnaiyan.

Subject of Research: Long-read nanopore sequencing to resolve hidden cancer-driving genetic alterations and a gene therapy-associated pediatric tumor

Article Title: Comprehensive DNA sequencing reveals hidden drivers of cancer

Article References: Comprehensive DNA sequencing reveals hidden drivers of cancer. Original publication (Bibliographic details unavailable.)

Image Credits: AI Generated

DOI: Not provided

Keywords: long-read sequencing, nanopore sequencing, cancer genomics, MiOncoSeq, precision oncology, BRCA2, haplotype phasing, gene therapy, adeno-associated virus, Krabbe disease, GLI1, tumor suppressor genes