The Landscape of microRNA Expression in HIV and Non-HIV Associated Classical Hodgkin Lymphoma through Next Generation Sequencing

Introduction

Classical Hodgkin lymphoma (cHL) is a lymphoma of B cell origin that affects both immune competent and immune suppressed patients. In this study, we sought to determine the complete landscape of microRNA expression in cHL, by performing deep sequencing of microRNAs in 66 patient samples. Further, we examined the associations of microRNA expression with clinical data, including HIV and EBV infection status, mixed cellularity and nodular sclerosis subtypes, and progression free and overall survival.

Methods

This cohort includes 66 cases of cHL of primarily mixed cellularity and nodular sclerosis subtypes. Nearly 50% of these cases were EBV positive and 39% were HIV positive. All the EBV(-), HIV(-) cases were nodular sclerosis subtype and nearly half of EBV(+), HIV(+) cases were mixed cellularity subtype.

From these cases, whole RNA was extracted from which small RNAs were selected via bead purification and subjected to next generation sequencing on the Illlumina platform. MicroRNA expression was assayed by mapping sequencing reads to the human genome and identifying those reads with matching sequences that were typical of a hairpin loop that characterizes microRNA precursors. We were able to identify 367 human microRNAs and 15 EBV microRNAs. The expression of these microRNAs was measured by normalizing the number of sequencing reads mapping to microRNAs within each case and across all the cases. Interestingly, we also found 18 novel microRNAs that have not been described previously in humans.

We tested the association of these microRNAs with progression-free and overall survival, as well as with histology, HIV and EBV status.

Results

We found a number of microRNAs that were robustly associated with stage. miR-138, miR-182, and miR-296 were associated with lower stage across all histologies, whereas miR-378 was strongly associated with higher stage.

We found that miR-92b, miR-138 and miR-186 were all associated with favorable prognosis with higher expression being associated with better outcomes.

We also found several microRNAs associated with histologic subtype. For example, miR-122 and miR-182 were highly expressed in nodular sclerosis cHL while miR211 was expressed highly in mixed cellularity cHL. miR-21 was highly expressed in all cases. EBV positive cases were defined in all tumors using in situ hybridization using an EBER probe. We found that expression of EBER was highly associated with EBV BART microRNAs, which were present in 100% of the EBV positive patients.  We found that miR-455 was highly expressed in HIV positive cases regardless of EBV status whereas miR-511 was expressed highly in all EBV cases in addition to EBV BART microRNAs.

Conclusion

Together, our data define the landscape of microRNA expression in HIV-associated and non-HIV-associated classical Hodgkin lymphoma and point to a role for microRNAs as novel biomarkers that distinguish histology, stage, outcome and EBV status.