A Circular RNA Molecule, circRAB11FIP1, Is Associated with TP53 Mutations and Is of Potential Prognostic and Functional Significance in Mantle Cell Lymphoma: Data from the Nordic MCL2 and MCL3 Studies

INTRODUCTION:

Circular RNAs (circRNAs) are covalently closed endogenous RNA molecules with tissue- and disease specific expression patterns, that are emerging as potential diagnostic and prognostic biomarkers. These molecules exert diverse regulatory functions, and several studies have reported prognostic relevance and functional significance of circRNAs in cancer. However, no studies have yet examined the role of circRNAs in Mantle Cell Lymphoma (MCL).

We have previously shown that the Nanostring nCounter technology enables specific, sensitive and accurate quantification of circRNAs in formalin-fixed, paraffin-embedded (FFPE) tissue samples from patients (Dahl et al, 2018). Here, we quantified circRNA expression in MCL tumors to address whether certain circRNAs could serve as prognostic markers in MCL. Samples were obtained from patients treated in the two Nordic clinical trials MCL2 and MCL3, all treated with immuno-chemotherapy and autologous stem cell transplant, which due to the improved prognosis observed with this regimen, remains the current standard of care.

MATERIALS AND METHODS:

We profiled the genome-wide circRNA expression in MCL using high-throughput RNA-sequencing (RNA-seq) of 14 diagnostic MCL samples, from which high-quality RNA from lymph nodes with MCL tumor infiltration was available. Based on these data, we designed assays for analyses of 41 differentially expressed circRNAs and quantified the expression using the NanoString nCounter technology. We examined the prognostic potential of individual circRNAs in a training cohort of 75 patients (MCL2) and confirmed these results in an independent, but similarly treated, validation cohort of 90 patients (MCL3).

RESULTS:

The total cohort consisted of 165 previously untreated MCL patients <66 years (median=58 [28-65]). There were no differences in baseline characteristics between the two cohorts, but significantly more patients in MCL2 were assigned to high risk groups according to MIPI-C than in MCL3. After a median follow-up of 11.4 years for MCL2 and 7.5 years for MCL3, the median time to progression (TTP) was 8.5 and 7.7 years, respectively.

In our training cohort we identified seven circRNAs that were significantly associated with both progression-free survival (PFS), TTP, overall survival (OS) and lymphoma specific survival (LSS). We dichotomized expression values of each individual circRNA and examined the prognostic value in the validation cohort MCL3. Only one (circRAB11FIP1) of the seven circRNAs identified in MCL2 retained prognostic value in MCL3. As shown in Figure 1, univariate analysis revealed that low circRAB11FIP1 expression was significantly associated with TTP in MCL2 (HR=5.1, [2.5;10.4], p<0.0001), while the effect in MCL3 was less pronounced, albeit significant; TTP (HR=2.8, [1.4;5.8], p<0.01). The same results were shown for LSS and PFS in both cohorts, but low circRAB11FIP1 was only significantly associated with OS in MCL2, not in MCL3.

Finally, we performed a multivariate cox regression analysis of the entire cohort and found that circRAB11FIP1 was an independent prognostic factor for TTP, even when adjusting for MIPI and ki67-index (HR=2.36[1.3;4.4], p<0.01). Interestingly however, we found an association between circRAB11FIP1 expression and presence of TP53 mutations (unpaired t-test, p<0.01). In patients without TP53 mutations, circRAB11FIP1 levels were significantly higher (mean=378.9[353.2;404.6] than in patients with mutations (mean=297.9[246.8;349.0]). When incorporating presence of TP53 mutations into the multivariate analysis, low circRAB11FIP1 was no longer an independent prognostic factor for TTP (HR=1.48[0.8;2.9], p=0.26).

CONCLUSION:

Our study showed that low circRAB11FIP1 was significantly associated with shorter TTP, even when adjusting for known prognostic factors, indicating that circRAB11FIP1 could play a specific role in the pathogenesis of MCL. CircRAB11FIP1 originates from exon 2 of the host gene RAB11-Family Interacting Protein 1 located on chromosome 8, and no studies have yet examined the functional role of this circRNA. Interestingly, we found that low circRAB11FIP1 was significantly associated with the presence of TP53 mutations. This warrants future studies to examine whether there is a functional link between circRAB11FIP1 and TP53 in MCL, which may help explain why patients with TP53 mutations have an exceedingly poor prognosis.

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