Photo by Darren Baker
A new technique can quickly and easily differentiate the two major subtypes of diffuse large B-cell lymphoma (DLBCL), researchers have reported in The Journal of Molecular Diagnostics.
The method is based on a reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) assay and 14 gene signatures.
It proved as accurate as the gold standard technology for identifying germinal center B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL.
So the researchers believe this RT-MLPA-based assay could help clinicians evaluate a patient’s prognosis and choose the optimal therapy for a person with DLBCL.
“Differences in the progression of the disease and clinical outcomes can, at least in part, be explained by the heterogeneity of lymphoma, which can be classified into two major subtypes with different outcomes,” said study author Philippe Ruminy, PhD, of the University of Rouen in France.
“Unfortunately, these lymphomas are morphologically undistinguishable in routine diagnosis, which is a major problem for the development of targeted therapies. Furthermore, array-based gene expression profiling (GEP), which is considered the gold standard for discriminating these tumors, remains poorly transposable to routine diagnosis, and the surrogate immunohistochemical (IHC) algorithms that have been proposed are often considered poorly reliable.”
With this in mind, Dr Ruminy and his colleagues tested the RT-MLPA-based assay alongside GEP techniques in samples from 259 patients with de novo DLBCL.
The team used a series of 195 patients from a single institution to train and validate the new technique. Of these patients, 115 had a classification determined with a Veracode DASL assay, 100 had an IHC classification, and 135 had survival data. A second series of 64 patients that were previously analyzed by Affymetrix U133+2 GEP arrays served as an external validation cohort.
When the researchers looked at 50 patients randomly selected from the training cohort, they found that the RT-MLPA-based assay classified 90% of the cases within the expected subtypes (20 GCB and 25 ABC). The remaining 10% (1 GCB and 4 ABC) were considered unclassified.
In the external validation cohort, the RT-MLPA-based assay classified 80% of cases within the expected subtypes (24 ABC and 28 GCB). However, 13.8% were considered unclassified (3 GCB and 6 ABC), and 4 were misclassified.
The researchers also found the RT-MLPA-based assay was sensitive for analyzing archived formalin-fixed, paraffin-embedded (FFPE) tissue samples. A comparison of samples from paired frozen and FFPE biopsies showed that the technique correctly classified 89.3% of 28 cases.
“Because RT-MLPA requires only short cDNA fragments for the correct binding and ligation of the gene-specific oligonucleotide probes, it is less affected by the use of low RNA concentrations and RNA degradation,” Dr Ruminy said.
“It could thus be used for the retrospective analysis of archival collections and for the inclusion of patients in prospective clinical trials, because only a few institutions routinely collect frozen biopsy material.”
To evaluate the prognostic value of the assay, the researchers looked at survival in 135 treated DLBCL patients who were diagnosed between 2001 and 2011.
Patients determined to have the ABC subtype by the RT-MLPA-based assay had significantly worse progression-free survival and overall survival than patients with the GCB subtype. And the expression of several individual genes within the MLPA signature was significantly associated with prognosis (ie, high LMO2, high BCL6, and low TNFRSF13B expression).
“The robust and cost-effective RT-MLPA assay can yield results within one day and requires reagents costing less than $5 per sample,” Dr Ruminy said. “Since RT-MLPA utilizes materials and equipment that are standard in many laboratories, the process can easily be implemented for routine use.”
