Comprehensive Analysis Of Microbial Signatures For Lymphomagenesis Using a Novel Microbial Detection Array

Infectious agents are estimated to play a causative role in approximately 20% of cancers worldwide. Viral infections, notably the Epstein-Barr virus (EBV) are associated with 10-15% of B-cell lymphomas and are found at a higher frequency in immunosuppressed patients with B-cell malignancy. The risks posed by these pathogens increases with age. Early detection can impact treatment options and may play a critical role in patient survival. We report the use of the Lawrence Livermore Microbial Detection Array (LLMDA), a highly sensitive and comprehensive detection system that contains probes for all known sequenced viruses and bacteria designed to identify pathogen-associated diseases. Our study focuses on detecting pathogens that may be associated with lymphomas, in order to develop appropriate therapeutic treatment strategies for pathogen-associated B-cell lymphomas.

The LLMDA is a pan-Microbial Detection Array (MDA) capable of detecting all known viruses (including phages), bacteria and plasmids and uses a novel statistical analysis method to identify organisms from complex mixtures hybridized to the array. The LLMDA contains a more comprehensive bacterial and viral target spectrum, more probes per target and is based on more updated sequence data than other existing microbial detection/discovery arrays. Family-specific probes representing complete genomes of all sequenced bacteria and viruses, conserved segments of these genomes, and plasmids were selected. Probes possess adequate sequence variation to allow detection of divergent species with homology to sequenced organisms. Using the LLMDA we tested indolent and aggressive stage B cell lymphomas and normal tissues as controls. We demonstrated the system’s accuracy by investigating the pathogen profiles of previously analyzed post-transplant lymphoproliferative disorder (PTLD) tumors. We also applied the technique to fresh frozen and formalin fixed, paraffin-embedded (FFPE) tissues to evaluate the range of sample types amenable to the LLMDA analysis.

We evaluated tissue from 34 lymphoma cases using LLMDA. These included 30 B cell (9 indolent and 21 aggressive stage), 2 T cell and 2 NKT cell, 4 plasmacytomas as well as 8 specimens of benign lymphoid tissue. Five of 21 aggressive stage B cell lymphomas were EBER+, and all the indolent stage B cell lymphomas were EBER-. Both NKT cell lymphomas were EBER+, and the T cell lymphomas and benign tissues were EBER-. Five of the PTLD cases (4 B cell lymphomas and 1 NKT cell lymphoma) were EBER+ and 5 were EBER-. Finally, two of the FFPE tissues (1 B cell lymphoma and 1 NKT cell lymphoma) were EBER+ and 1 EBER-, in conformity with the previously analysis of EBER status in these samples. We have confirmed the accuracy of the technique by detecting EBV in EBV-positive lymphomas while observing no false-positive results in EBV-negative lymphomas

We have confirmed the LLMDA’s efficacy for detecting viruses in pathogen-associated disease. This technique may provide a powerful and sensitive method for identifying known viral pathogens associated with tumors and may also prove useful for the discovery of novel tumor-associated viruses.

No relevant conflicts of interest to declare.