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Columbia Launches Clinical Cancer Exome and Transcriptome Test


NEW YORK (GenomeWeb) – A clinical cancer whole-exome and transcriptome sequencing test recently launched by Columbia University's Laboratory of Personalized Genomic Medicine promises to impact the treatment of cancer patients.

The new test was developed as part of a wider effort to incorporate personalized medicine approaches into Columbia's pediatric oncology program, according to Andrew Kung, chief of the division of pediatric hematology, oncology, and stem cell transplantation.

With the rapid development of next-generation sequencing technology and decrease in sequencing costs, "the feeling was that three to five years from now, instead of looking at panels for specific mutations, looking at the whole genome will be the way that cancer medicine will be practiced," Kung told Clinical Sequencing News. "That's why we set off to develop a comprehensive approach for looking for changes in cancer genomes."

The test is currently available to pediatric oncology patients with a high risk of relapse. The plan is to scale up capacity to be able to offer the test, which requires frozen tumor, to all pediatric cancer patients by the end of this year, and to adult cancer patients eventually.

To complement the test, the Laboratory of PGM has also been developing a targeted cancer panel that includes more than 300 well-established cancer genes. That assay, which has been validated in paraffin-embedded tissue with as little as 50 nanograms of input DNA, is expected to come online in July.

Since the beginning of this year, 17 patients have undergone the cancer exome and transcriptome test. In about 30 percent of cases, physicians have made a clinical decision based on the results, Kung said. In some cases, the test detected a genetic alteration for which a targeted therapy was available, while in others, it prevented the use of a targeted drug because a mutation was absent.

In at least four cases, the test revealed information that no existing targeted test would have uncovered, Kung said.

In the case of one patient with relapsed leukemia, for example, the test found a mutation in a receptor tyrosine kinase that is not normally associated with leukemia and would not have been detected by a targeted leukemia-specific test. The child received a targeted therapy and "had a very impressive clinical response," Kung said.

The new test runs in the Laboratory of PGM's CLIA lab that is also accredited by the College of American Pathologists, CAP, and the New York State Department of Health's Clinical Laboratory Evaluation Program, CLEP.

The price of the test has not been set and might vary depending on what samples are analyzed, but will likely be in the range of $6,000 to $10,000, according to Peter Nagy, one of the laboratory's directors. Internal funding will support the test for the time being to be able to demonstrate its clinical value, but in the long term, it is expected to become a "full reimbursable test," Kung said.

The test builds on a germline exome sequencing test for inherited diseases that the lab launched last year. Like that test, it uses an Agilent exome capture kit, followed by sequencing on the Illumina HiSeq 2500.

The new test requires a blood sample for the control exome and a frozen tumor sample for the cancer exome and transcriptome. In some cases, several tumor samples from different disease stages may be included. The tumor samples are dissected in order to have at least 50 percent tumor content, which is important for detecting copy number variants.

According to Nagy, a big part of the development was to devise a process for getting the tumor sample from the operating room to the laboratory quickly.

Both the tumor and germline DNA are sequenced to greater than 150-fold average coverage, which ensures that about 98 percent of the target regions are covered at least 10-fold, Nagy said.

For the tumor transcriptome, the sample is depleted of ribosomal RNA, and at least 50 million uniquely mapping high-quality reads are generated per sample.

The germline exome is analyzed for mutations in cancer-predisposing genes, mutations in pharmacogenomic genes, and mutations in the 56 genes recommended by the American College of Medical Genetics and Genomics if patients indicate in the consent form that they would like those secondary findings to be included.

In the tumor exome, more than 600 genes that have been associated with cancer in the literature are analyzed for tumor-specific somatic mutations, including copy number changes. In addition, patients are consented to allow researchers to study mutations in other genes later on.

By focusing the clinical analysis on 600 genes, which include all "actionable" mutations that can be targeted with drugs, results can be returned within four weeks, a timeframe that allows clinicians to consider them in therapy decisions, Kung explained, though not all cases so far have required this short turnaround time.

In the transcriptome data, the lab looks for gene fusions, highly overexpressed genes, or non-expressed genes. It also uses those data to check whether mutated genes found in the tumor exome are actually expressed.

A list of relevant variants is submitted to a molecular tumor board, which includes oncologists, where they are further reviewed and their significance for the patient is evaluated. Based on that, the variants are placed into four tiers: clinically actionable variants known for the patient's tumor type; strongly cancer-associated or actionable variants known from other tumor types; other variants in cancer-associated genes; and variants of unknown significance.

Mutations in the first tier are confirmed by Sanger sequencing or another targeted method, and a report is issued to the treating oncologist.

The lab has extensively validated the test on a set of frozen tumor samples and controls. It has been "very robust in terms of being able to get all the information we're looking for," Kung said, having missed no major alterations that would have impacted therapy. However, more cases will need to be analyzed to determine the sensitivity and specificity of the test, he said.

"We make it very clear to the clinicians that this is a screening test," Nagy said. "We do not say that we will find every mutation and every translocation, and every copy number change that is relevant to the patient's condition."

One current weakness of next-generation sequencing in general is the detection of insertions or deletions 50 to 500 bases in size, he said, but only a few genes with variants of that type that impact cancer treatment are known.

The lab has submitted the new test for approval to the New York State Department of Health. Based on its experience with previously submitted tests, including its germline exome test, submitted in early 2013, and an Illumina TruSeq targeted cancer panel, submitted about five months ago, it does not expect a decision soon – neither test has been approved yet. While a test is under review, laboratories can request waivers for individual patients to administer the test, which the department usually grants, Nagy said.

One difference between Columbia's new cancer exome and transcriptome test and cancer exome tests offered by others is "the intense focus on the workflow around the assay that enables us to use this as a clinical test," Kung said. He and his colleagues paid a lot of attention to developing educational material for patients to be able to provide the proper informed consent, he said, developed a workflow that enables them to return results in a clinically timely manner, and generated reports that convey the information necessary for a clinician to make an informed decision. This, he said, "may be a little different than other centers that are simply doing a lot of sequencing for research purposes."

The Columbia team is currently studying the clinical utility of the test, using patient-derived xenograft models, which they generate for each patient where sufficient tumor material is available. Results from those experiments are not used to inform treatment decisions for the patient, though, because they take too long to obtain.

This xenograft approach involves testing not only treatments that are suggested by the exome and transcriptome sequencing test in the mouse model but also therapies that are not predicted to work. "Only with this two-pronged approach can you really start asking the question of what's the positive or negative predictive value of [the test's] findings," Kung said.