Prognostic Significance Of a 4-Microrna Signature Targeting JAK2 In Classical Hodgkin Lymphoma

The behavior of classical Hodgkin lymphoma (cHL) is determined by both the intrinsic features of the Hodgkin/Reed Sternberg (HRS) cells and the characteristics of the microenvironment, making the analysis of entire lymph nodes an effective approach to understanding the disease. One of the genetic lesions most frequently found in HRS cells involves members of the JAK/STAT signaling pathway. However, few studies have identified microRNAs (miRNAs) that target JAK2 in cHL. In a previous study, our group identified miR-135a as a JAK2 regulator and showed the prognostic impact of this miRNA in a small set (n=89) of cHL patients (Navarro A. et al. Blood 2009). In the present study, we have examined novel miRNAs targeting JAK2 and evaluated the prognostic impact of their expression levels in 170 lymph nodes of cHL patients.

TargetScan and miRbase were used to identify JAK2-related miRNAs. The conserved miRNAs with higher scores were selected and further validated by Renilla-Luciferase assay and Western Blot. We performed a Renilla-Luciferase assay with a modified expression vector psiChecK-2 containing the complete 3’UTR region of JAK2 cloned in the 3’UTR region of Renilla luciferase gene. For Renilla-Luciferase assay, 100nM of the pre-miRNAs of interest or pre-miR negative control were transfected in the HDMYZ cHL cell line together with 1μM of modified psicheck2 vector using Lipofectamine 2000, and luminescence was read at 24 hours. Further validation of the miRNA target was performed by Western Blot in three cHL cell lines (L428, L-1236 and HDMYZ). The expression levels of the miRNAs identified as targeting JAK2 were then assessed in lymph nodes from 170 cHL patients (median age, 33 years; male, 51%; EBV, 43.8%; HIV, 12%) diagnosed and treated in a single institution. Moreover, 15 reactive lymph nodes (RLN) were used as normal controls. RNA was purified from formalin-fixed paraffin-embedded lymph nodes using RecoverAll Total Nucleic Acid Isolation kit. The miRNA expression was analyzed using TaqMan microRNA assays. Statistical analyses were performed using R v2.13 and PASW Statistics 18.

The bioinformatic analysis identified seven microRNAs as possible regulators of JAK2 (miR-34a, miR-101, miR-135a, miR-204, miR-216a, miR-216b and miR-375). The Renilla-Luciferase assay confirmed that in addition to miR-135a, miR-101 (61%; p=0.01), miR-204 (46%; p=0.003) and miR-216a (64.8%; p=0.02) also targeted JAK2. These findings were confirmed by Western Blot analysis of JAK2 levels after transfection with pre-miRNAs (median % of protein reduction in the 3 cell lines of 21.1%, 46.7% and 24%, respectively). The analysis of these miRNAs in RLN showed that miR-101, miR-135a and miR-204 were significantly downregulated in lymph nodes of cHL patients (p<0.001, p<0.001 and p=0.02 respectively) in comparison with RLN used as control. In contrast, miR-216 did not show significant differences in comparison with RLN. Interestingly, the analysis of the expression levels of these 4 miRNA in the patient lymph nodes had an impact on prognosis. First, low expression levels of miR-135a was associated with lower rate of complete response to first line treatment (76% vs 89%, p=0.012) and a trend was observed for miR-216(p=0.077). Secondly, patients with low expression levels of miR-101 or miR-204 had shorter disease-free survival (DFS) (151 vs 109 months, p=0.020; and 149.2 vs 105.5 months, p=0.055). Finally, patients with low levels of miR-135a, miR-204 or miR-216 had shorter overall survival (OS) (158 vs 136 months; p=0.039; and 157.5 vs 110 months, p=0.025; and 165 vs 140 moths, p=0.011 respectively). Since all four miRNAs showed prognostic impact (DFS, OS or treatment response), we decide to analyze the prognostic impact of the combination of the 4 miRNAs. Patients were then classified in two subgroups according to the expression levels of all four miRNAs. Patients with low expression of more than 2 miRNAs had shorter OS (163 vs 132 months; p=0.012). The multivariate analysis identified the combination of the four miRNAs as an independent prognostic marker of OS (OR, 7.6; 95% CI, 1.1-57.2; p=0.048) together with Age≥45years (OR, 5.9; 95% CI, 2.4-14.4; p<0.001) and B symptoms (OR, 2.6; 95% CI, 1.09-6.3; p=0.03).

MiR-101, miR-135a, miR-204 and miR-216 regulate JAK2 and are independent prognostic factors in cHL.

SDCSD of School of Medicine of UB. AC is an APIF-UB fellow.

No relevant conflicts of interest to declare.