Comprehensive DNA sequencing reveals hidden drivers of cancer

Michigan’s Oncology Sequencing Program, or MiOncoSeq, was the first of its kind in the nation to sequence patient tumors.

Launched in 2010, the program analyzes DNA and RNA from normal tissue and tumors to identify alterations that could help guide treatment.

In a Cancer Discovery study, U-M researchers have enhanced the process with long-read nanopore sequencing, which can detect alterations that might be missed by conventional sequencing.

Conventional short-read sequencing examines small pieces of DNA, typically a few hundred DNA letters long.

Long-read sequencing can read thousands to tens of thousands of letters in a single stretch, allowing researchers to see complex changes and how they fit together.

“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.”

Most genes come in two copies, one inherited from each parent.

Nanopore sequencing can help clinicians understand whether one or both copies of a gene that protects against cancer are disabled.

This can occur through complex DNA rearrangements and chemical modifications.

Having a list of mutations doesn’t always explain a tumor. We need to understand how these mutations are arranged, which conventional sequencing often cannot resolve.” 

-Arul Chinnaiyan, M.D., Ph.D., S.P. Hicks Professor of Pathology and Urology

It can also show whether multiple mutations occur together on a single copy of a cancer-promoting gene, potentially increasing its activity or contributing to drug resistance.

In one arm of the 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.

In a broader analysis, the researchers screened 4,496 tumor samples from 3,688 patients in the MiOncoSeq program.

They then used 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 team showed how long-read sequencing can also uncover inherited cancer risk missed by conventional testing.

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.

Tracing the origin of a rare pediatric tumor

In a separate report in the New England Journal of Medicine, the team used nanopore sequencing to help explain how a rare tumor developed in a 3-year-old boy after gene therapy.

The patient 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 after 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 gene therapy, routine imaging revealed a small tumor beside his spine.

Through nanopore sequencing, the team found that a fragment of DNA from the modified AAV had inserted into the GLI1 gene.

The 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.”

Additional authors: 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.

Tech transfer(s)/Conflict(s) of interest: National Cancer Institute Prostate SPORE Grant (P50-CA186786), NCI Early Detection Research Network (U2C-CA271854), NCI Research Specialist Award (NCI 50CA293826), and NCI Outstanding Investigator Award (R35-CA231996, to A.M. Chinnaiyan).

Papers cited:

“Long-Read Haplotype Phasing Resolves Allelic Configuration as a Missing Layer of Precision Oncology” Cancer Discovery. DOI: 10.1158/2159-8290.CD-26-0839

“Integration-Associated Paraspinal Neoplasm After AAV Gene Therapy,” New England Journal of Medicine. DOI: 10.1056/NEJMc2607927

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