Senior Staff Writer

Children's Hospital Los Angeles (CHLA) cares for roughly 20% of the nation's retinoblastoma cases, seeing up to 50 new patients a year. But to test for retinoblastoma, a malignant cancer of early childhood that can start growing at any time before birth up until about age seven, pathologists at CHLA had to send samples out to a lab in Toronto and wait anywhere from four to 10 weeks to get the results back.

To address this issue, CHLA researchers developed a unique next-generation sequencing test for the gene linked to retinoblastoma. The new approach is much more comprehensive, said Maurice O'Gorman, chief of laboratory medicine in the department of pathology and lab medicine at CHLA. O'Gorman told Medical Device Daily that the new test covers essentially the entire gene from beginning to end, whereas the classic method only sequenced specific hot spots, or parts of the gene previously associated with mutations that lead to retinoblastoma.

Not only is the in-house test more comprehensive, clinicians can have the results back in days rather than weeks or months, O'Gorman said. And early detection of the cancer's genetic cause makes a big difference in treatment outcomes.

Retinoblastoma arises from immature retinal cells in one or both eyes. Among infants and children, common signs of this cancer include having a white glow or glint in the pupil of one or both eyes, the presence of a white pupil in a color photo, or crossed or misaligned eyes, according to CHLA. When left untreated, retinoblastoma can result in blindness or death. In germline cases, the disease causing mutation is present in cells throughout the body resulting in tumors in both eyes, as well as in other locations throughout the body.

The retinoblastoma next generation (RB1 NextGen) sequencing panel was developed based on deep DNA sequencing technology, using new technical and bioinformatics methodology to sequence the entire RB1 gene.

"Typically, we have used NextGen sequencing capability to sequence many genes at a relatively low depth of coverage," said Alexander Judkins, MD, head of the department of pathology and laboratory medicine at CHLA. "Our team took a very different approach by deciding to look at a high depth of coverage of a single gene, sequencing through the entire gene, which is technically very difficult to do."

With the existing NextGen sequencing approach for retinoblastoma, there are regions of genes that do not get examined at all, potentially allowing significant data to be missed, according to Timothy Triche, MD, PhD, director of the center for personalized medicine at CHLA. "New data tells us that the non-coding DNA is extremely important. And one of the strengths of our approach is in examining those non-coding areas."

By sequencing the whole gene, researchers are able to see more frequent occurrences of germline mutations of the RB gene. Because germline mutations can be passed on, this knowledge can better inform the correct treatment approach for patients when these mutations are identified, the researchers noted.

"We used to think that if a child had a tumor in one eye and not in the other eye, it was a result of a somatic mutation. And because somatic carriers don't pass on mutated genes, we believed removal of the tumor stopped the cancer," said Tom Lee, MD, director of the Vision Center at CHLA. "But now we know that there is a percentage, on the order of 10% to 15% of those children who had an apparent somatic mutation, who are actually carrying a germline mutation, putting them at risk of developing subsequent tumors during adolescence or early adulthood."

Lee said the ability to determine the existence of RB1 germline mutations early has significant implications, not only for the prognosis of a new patient with retinoblastoma, but for siblings who could also be at risk and eventually for a patient's own children, if a germline mutation is detected in a retinoblastoma survivor that could be passed on to future generations.

"Our clinicians are extremely excited," O'Gorman said. "We are putting in place mechanisms to allow them to expand access for all of the patients they are seeing and all of the families of the patients they have seen."

He added that the hospital developed the service to provide retinoblastoma testing for any child or family member in the U.S. who requires it. It's an important compliment to CHLA's history with this particular cancer gene, O'Gorman added, because the gene that causes retinoblastoma was discovered at CHLA in 1987. Instead of being an onco gene, the gene linked to retinoblastoma is considered a tumor-suppression gene, O'Gorman explained.

CHLA now has the ability to also offer this unique and comprehensive screening test to survivors of the disease or their family members, as well as to patients at other cancer centers. "We believe that siblings and children of carriers of the germline should be tested immediately after birth because detecting the gene mutations early can save a patient's vision," Lee said.

Roughly two-thirds of retinoblastoma patients at CHLA have unilateral cases of RB, or cancer in just one eye. While only 15% of cases will test positive for a germline mutation, physicians must treat all patients as though the cancer could potentially be bilateral, or present in both eyes, a condition commonly associated with having a germline mutation.

"This test enables our researchers to look at the RB1 gene more comprehensively," Judkins said. "The collection of new genomic data on RB1 will hopefully enable basic researchers to translate this material into biologically meaningful insights, leading to earlier detection and new treatments for both primary and recurrent disease."

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