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Inexpensive Assay Differentiates Two Diffuse Large B-Cell Lymphoma Subtypes

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NEW YORK (GenomeWeb) – Although it has been known for over a decade that two subtypes of diffuse large B-cell lymphoma (DLBCL), a form of non-Hodgkin lymphoma, have different prognoses and may respond differently to targeted treatment, routinely distinguishing between them has proved challenging.

Now, a team of researchers in France has developed an inexpensive classification method using a panel of 14 gene signatures and garden-variety lab equipment that may provide competition for commercial products being developed as companion diagnostics.

The method, published online in the Journal of Molecular Diagnostics yesterday, is based on assessment of publically available Affymetrix RNA data on the two DLCBL subtypes — germinal center B-cell-like (GBC) and activated B-cell-like (ABC).     

DLBCL accounts for up to 40 percent of non-Hodgkin lymphomas, and the American Cancer Society estimates there will be nearly 72,000 new non-Hodgkin lymphoma diagnoses in 2015.

Of the two DLBCL subtypes, "ABC is an aggressive disease, and GCB, while it's aggressive, has a better prognosis," Philippe Ruminy, an author on the JMD study and a researcher at the University of Rouen, told GenomeWeb.

Furthermore, mounting evidence suggests the subtypes will respond differently to molecularly targeted therapies. These should soon be available in the clinic and will likely be applicable specifically to one or the other subtype, so "It becomes increasingly important to be able to distinguish these pathologies," Ruminy noted.

The problem is, it's "nearly impossible" to reliably distinguish GBC from ABC, he said. Immunohistochemical techniques to do so are inexpensive, but there is "very bad reproducibility" between pathologists, Ruminy said. Molecular methods such as array-based gene expression profiling have stepped in, but they are time consuming and expensive, and so far none has been commercialized.

One example of a molecular technique, a DLBCL subtyping kit being developed by NanoString Technologies, is very similar to the JMD method, Ruminy said.

"Their kit may be more advanced than ours because they are already working in industry," he said. He pointed out, however, that the NanoString method requires that firm's platform, which he said is "pretty expensive, and to the best of my knowledge, for example, in France there is only one."

In contrast, the JMD method relies on reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA), capillary electrophoresis, and freely available software the group developed to classify resulting data.

After a standard RNA extraction step, the assay takes about one day, Ruminy said. "It's very fast, and also it works pretty well on FFPE samples." The reagent cost is less than $5 per sample.

In the study, Ruminy and his colleagues used the 14-gene signature to classify a validation cohort of 46 samples, showing the RT-MLPA method was equivalent to an Affymetrix gold standard.

The method was then validated using an independent cohort of 64 frozen patient samples and paired FFPE samples from 30 of these patients. RT-MLPA classified about 84 percent of the frozen samples and 89 percent of the FFPE samples within the expected subtypes.

In a cohort of 135 patients, the group then confirmed overall survival and progression-free survival differences between the two subtypes.

Ruminy noted that the NanoString method does "exactly the same thing" as the RT-MLPA technique. He presented his group's work in practically the same time slot but a different session as a presentation on the NanoString-based assay at the 2013 American Society of Hematology meeting. 

NanoString is now developing its method as a companion diagnostic for Celgene's Revlimid (lenalidomide) to treat DLBCL. The firm recently told GenomeWeb, "Over time companion diagnostic partnerships will become a significant driver of growth and cash flow for our business."

The firm is also working with two as-yet-unnamed pharma companies. Analysis from researchers at AstraZenaca, published in Clinical Cancer Research, recently concluded "DLBCL segmentation of patient tumor samples is possible using a number of expression platforms … however, we found that NanoString offers the most flexibility and fewest limitations in regards to robust clinical tissue subtype characterization."

AstraZeneca's anti-CD19 monoclonal antibody drug MEDI-551 is currently in a few clinical trials, including ones for B-cell malignancies.

Ruminy pointed out another drug, ibrutinib, that may potentially become a targeted therapy for DLBCL subtypes. Branded Imbruvica by drug sponsor Pharmacyclics, ibrutinib was recently approved by the US Food and Drug Administration for a subset of chronic lymphocytic leukemia patients. There are currently also a number of ongoing clinical trials of ibrutinib for DLBCL.

As for the RT-MLPA method, Ruminy's group has filed a patent. "But for the commercialization, we are just a research team, so we don't have the ability to develop or to sell diagnostic kits," he said, adding, "If we could find an industrial partner that would be interested in this, it would be great." 

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