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NGS Shows Potential for Disease Monitoring and Diagnosis of Cutaneous T-Cell Lymphomas

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In a study using the Illumina HiSeq, researchers from Stanford University have demonstrated that high-throughput sequencing can be used to identify and monitor residual disease in patients with certain forms of cutaneous T cell lymphoma to better assess their response to treatment and determine whether they are in remission.

The effort, which the group described in a paper in Science Translational Medicine earlier this month, found that a sequencing-based approach could be a more sensitive diagnostic technique for mycosis fungiodes and Sézary syndrome — two forms of cutaneous T cell lymphoma — than currently used methods combining immune histopathology and PCR-based clonality measurements.

Wen-Kai Weng, the study's first author told Clinical Sequencing News that the team's hope is that the technique can be used to precisely track patients' molecular status after treatment as a way to potentially predict future recurrence and enable interventional treatment earlier.

According to Weng, the lack of a sensitive test to identify and monitor disease in these lymphomas was not as much of a problem previously because few effective therapies were available.

More recently though, he said, allogenic hematopoietic cell transplant has proved to be an effective treatment for some MF/SS patients. Because of this, monitoring of disease post-transplant is looking like more of a critical area of need.

"In the past, it was 'so what, we have a bad test, it doesn’t matter,'" he said. "But now we have better treatment, so there is more interest in having a test that can determine how many people really get a good remission."

The group believes high-throughput sequencing could help clinicians predict which patients are in a stable remission and which are likely to recur, enabling them to change their treatment strategy to prolong remission, for example, by reducing immunosuppressive therapy or adding other biologic therapies in patients who show molecular signs that they are going to recur, the study authors wrote.

According to the study authors, while flow cytometry and PCR have been the main tools available for diagnosing and monitoring these cancers previously, they have not proved to be very powerful.

Flow cytometry is problematic because malignant cells in these lymphomas are not characterized by unique surface protein expression patterns. Up to 10 percent of skin or blood cells can show the same surface marker as malignant clones. "So unless you have a really high percentage of cancer cells, you can't tell," Weng said.

PCR, meanwhile, lacks sensitivity and can be confounded when PCR products representing non-malignant clones are detected together as one peak, effectively mimicking the presence of a true malignant clone.

"With high-throughput sequencing we are actually looking at the specific unique clone sequence. Once we identify that, later on, we can use it to track whether there is disease in the blood or skin after treatment," Weng said.

"This idea is not novel, he added, but with [sequencing] technologies developing to the point where we can use it at a reasonable price and reasonable speed, it made sense to try this now."

In the ongoing study, Weng and his colleagues are sequencing the T cell receptors in patients with Sézary syndrome before and after they receive allogenic hematopoietic cell transplants.

The group's recent publication represents 10 of the first patients analyzed, but Weng said that the group has now sequenced 29 patients in total.

In the study, the group sampled both peripheral blood mononuclear cells and skin cells, and then amplified extracted DNA using sequence-specific primers to generate up to 1,000,000 reads spanning the CDR3 region of individual cells. They then identified malignant clones based on the presence of a dominant CDR3 sequence.

The group also performed experiments using blood samples spiked with purified cancerous cells to demonstrate that the method could detect these cells at concentrations as low as 1 in 50,000 PBMCs — a sensitivity significantly greater than that of current diagnostic methods.

Of the 10 patients the researchers analyzed, nine had clinical evidence of active disease prior to transplant, but only four had circulating cancer cells identified using flow cytometry. However, when the researchers performed sequencing on the six samples with no detectable cancer cells by flow cytometry, they measured the presence of malignant clones at between 0.35 and 0.58 percent of the total TCR sequences.

After treatment, sequencing results also correlated with remission and recurrence outcomes, a promising hint at the method's potential clinical utility.

In other forms of lymphoma, Weng said, research has shown that molecular measurements of recurrence can predict future clinical recurrence up to six months later. This was also true for one patient in the Stanford group's cutaneous T cell lymphoma study, who showed increases in malignant sequences four months before having a clinical recurrence.

In addition to continuing the current study, Weng said he and his colleagues are also planning to study whether high-throughput sequencing might be able to aid in the diagnosis of these lymphomas up front, which can be difficult using standard clinical assessments.

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