Sequencing cancer mutations - there's an app for that

February 17, 2012

Using precise information about an individual’s genetic makeup is becoming increasingly routine for developing tailored treatments for breast, lung, colon and other cancers. But techniques used to identify meaningful gene mutations depend on analyzing sequences of both normal and mutant DNA in tumor samples, a process that can yield ambiguous results. Now, a team of Johns Hopkins researchers says it has developed an easy-to-use online computer software application that can clear up any confusion faster and cheaper than other methods currently used to do the job.

The application, called “Pyromaker,” is available free-of-charge at , and a related tutorial will be posted there soon. The software generates simulated pyrograms, which are readouts from a gene sequencing technique known as pyrosequencing.

Most pyrograms correspond precisely to a person’s unique mutation or set of mutations, but some mutations can be more difficult to interpret than others, the Johns Hopkins researchers say.

“Pyromaker’s value is in rapidly sorting through each of several simulated pyrograms, until there is a clear match with the actual tumor pyrogram,” says James R. Eshleman, M.D., Ph.D., a professor in the departments of Pathology and Oncology at Johns Hopkins. “Pyromaker enables us to do in minutes, essentially at no cost, what otherwise would take days of further, expensive tests.”

Eshleman led the team that developed Pyromaker, the software code for which was written by Johns Hopkins pathology resident Matthew T. Olson. A report on the application, with demonstrations of Pyromaker’s ability to resolve sequencing ambiguities, was published online in the Journal of Molecular Diagnostics.

Swedish researchers invented pyrosequencing in the mid-1990s as an alternative to the traditional method of gene sequencing. Pyrosequencing is an automated process in which solutions of the four DNA-building nucleotides, or “bases” (G, C, T, A) are added, one at a time, to single strands of DNA from sampled cells, in order to build up a complementary matching strand. A match with the next available base causes a chemical reaction with a special enzyme, which in turn emits a light pulse, resulting in a “peak” on the pyrogram – effectively indicating the next base in the DNA sequence.

Pyrosequencing works on shorter stretches of DNA than does the traditional method, known as “Sanger,” named for Frederick Sanger who invented the process.  But pyrosequencing is also more sensitive in registering the presence of mutant DNA in a tumor sample, which is a mix of tumor and normal cells. That sensitivity makes it very useful for tumor sequencing, says Eshleman, because the mutant genes that drive a tumor’s abnormal growth typically are less prevalent in a tumor sample, compared with normal versions of those genes.

Because a tumor pyrogram is an overlay of both healthy and mutant DNA, identifying the correct sequence may be difficult and further studies to sort it all out can delay diagnosis and add significantly to costs, he says.

To develop the software, Eshleman and his team first confirmed that simulated pyrograms generated by Pyromaker matched actual pyrograms of tumor DNA containing well-known, common cancer-driving mutations in the genes KRAS, BRAF, GNAS and p53. Then, they focused on the example of KRAS, an “oncogene” that helps drive many types, to show that nearly all one- and two-base mutations in KRAS have distinguishable pyrograms.

In a few cases, the researchers found that the pyrograms of two different mutations appear identical under certain sequencing conditions. With Pyromaker, they were able to show that pyrograms of two such mutations would become readily distinguishable if they were re-sequenced with a different order of added bases.

Finally, the researchers applied Pyromaker to ambiguous KRAS pyrograms from two colorectal cancer patients, and compared the results with tests using Sanger sequencing and other relatively time-consuming methods to resolve the ambiguities. The comparison showed that Pyromaker could resolve the ambiguities more quickly.

Eshleman notes that Pyromaker also may be used with a new sequencing technology known as ion semiconductor sequencing, which detects a change in hydrogen ions instead of a light pulse.

The software was written by Guoli Chen and Matthew T. Olson, both from Eshleman’s laboratory in the Department of Pathology at Johns Hopkins. Other scientists who contributed to the research include Alan O’Neill, Alexis Norris, Katie Beierl, Shuko Harada, Marija Debeljak, Keila Rivera-Roman, Samantha Finley, Amanda Stafford, Christopher David Gocke, and Ming-Tseh Lin, all from Johns Hopkins.

Contributions to funding the research came from the Sol Goldman Pancreatic Cancer Research Center, the Michael Rolfe Foundation, the Dick Knox Foundation, and the National Institutes of Health (CA130938).

Explore further: New virtual tool may provide more accurate diagnosis of genetic mutations

Related Stories

New virtual tool may provide more accurate diagnosis of genetic mutations

February 6, 2012
DNA sequencing to detect genetic mutations can aid in the diagnosis and selection of treatment for cancer. Current methods of testing DNA samples, Sanger sequencing and pyrosequencing, occasionally produce complex results ...

Slow growth of childhood brain tumors explained

June 23, 2011
(Medical Xpress) -- Johns Hopkins researchers have found a likely explanation for the slow growth of the most common childhood brain tumor, pilocytic astrocytoma. Using tests on a new cell-based model of the tumor, they ...

Scientists show how BRCA1 cancer gene mutations harm breast cells

December 14, 2011
(Medical Xpress) -- Working with human breast cells, researchers at the Johns Hopkins Kimmel Cancer Center have shown how the inactivation of a single copy of the breast cancer gene BRCA1 leaves breast cells vulnerable to ...

Elusive gene mutations found for malignant brain tumor

August 4, 2011
A discovery by scientists at Duke University Medical Center and Johns Hopkins University could increase the chances for an effective combination of drug therapy to treat the second most common type of brain tumor.

Recommended for you

Shooting the achilles heel of nervous system cancers

July 20, 2017
Virtually all cancer treatments used today also damage normal cells, causing the toxic side effects associated with cancer treatment. A cooperative research team led by researchers at Dartmouth's Norris Cotton Cancer Center ...

Molecular changes with age in normal breast tissue are linked to cancer-related changes

July 20, 2017
Several known factors are associated with a higher risk of breast cancer including increasing age, being overweight after menopause, alcohol intake, and family history. However, the underlying biologic mechanisms through ...

Immune-cell numbers predict response to combination immunotherapy in melanoma

July 20, 2017
Whether a melanoma patient will better respond to a single immunotherapy drug or two in combination depends on the abundance of certain white blood cells within their tumors, according to a new study conducted by UC San Francisco ...

Discovery could lead to better results for patients undergoing radiation

July 19, 2017
More than half of cancer patients undergo radiotherapy, in which high doses of radiation are aimed at diseased tissue to kill cancer cells. But due to a phenomenon known as radiation-induced bystander effect (RIBE), in which ...

Definitive genomic study reveals alterations driving most medulloblastoma brain tumors

July 19, 2017
The most comprehensive analysis yet of medulloblastoma has identified genomic changes responsible for more than 75 percent of the brain tumors, including two new suspected cancer genes that were found exclusively in the least ...

Novel CRISPR-Cas9 screening enables discovery of new targets to aid cancer immunotherapy

July 19, 2017
A novel screening method developed by a team at Dana-Farber/Boston Children's Cancer and Blood Disorders Center—using CRISPR-Cas9 genome editing technology to test the function of thousands of tumor genes in mice—has ...


Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.