FOXP1 Expression Is a Prognostic Biomarker in Follicular Lymphoma Treated with Immunochemotherapy and Is Associated with Distinct Molecular Features

Introduction: Follicular lymphoma (FL) is a clinically and molecularly highly heterogeneous disease, yet prognostication relies predominantly on clinical tools. We have recently demonstrated that integration of the mutation status of seven genes (including EZH2 and MEF2B) into a combined clinico-genetic risk model (m7-FLIPI) improves risk-stratification, and represents a promising approach to identify a subset of patients at highest risk of treatment failure. However, the mechanism underlying the association between mutations of the 7 genes included in the m7-FLIPI and outcome has thus far not been elucidated. We assessed whether downstream phenotypic commonalities of gene mutations predict outcome following immunochemotherapy and whether select genotypic and phenotypic alterations are associated with FOXP1 expression.

Methods: We mined gene expression data to uncover genes that are differentially expressed in EZH2 - and MEF2B -mutated FLs. We focused on FOXP1 and assessed its protein expression by immunohistochemistry (IHC) in a total of 763 tissue biopsies. For outcome correlation, a population-based training cohort of 142 FL patients treated with R-CVP, and a clinical trial validation cohort comprising 395 patients treated with CHOP +/- rituximab were used. To differentiate cases with high vs. low FOXP1 expression, we chose the cut-off that maximized the log-rank test statistic for failure-free survival (FFS) in the training cohort. The effect of FOXP1 expression on FFS in the validation cohort was estimated using Cox regression analysis. In addition, for exploratory analyses, we used specimens from 350 BC Cancer Agency patients, 124 of whom overlapped with the above-mentioned training cohort.

Results: We found FOXP1 to be significantly down-regulated in both EZH2 - and MEF2B -mutated cases. The cut-off value that best separated favorable from unfavorable FFS in the training cohort was determined to be >10% percent tumor cells expressing FOXP1 based on the log-rank test statistic. 76 specimens in the training cohort (54%) had high FOXP1 expression, which was associated with reduced 5-year failure-free FFS (55% vs. 70%, P=0.035). In the validation cohort, high FOXP1 expression status was observed in 248 patients (63%) and correlated with significantly shorter FFS in patients treated with R-CHOP (HR 1.95, P=0.017), but not in patients treated with CHOP (HR 1.15, P=0.44), demonstrating that FOXP1 is a predictive biomarker. In BCCA patients, high FOXP1 expression remained significantly associated with inferior FFS in a multivariate Cox regression model including rituximab maintenance (by intention to treat) and FLIPI high vs low/intermediate (HR 1.84, 95%-CI [1.10-3.08], P=0.021). Similarly, in the GLSG cohort, high FOXP1 expression was significantly associated with poor FFS in patients treated with R-CHOP, after adjusting for rituximab, FLIPI and the interaction of FOXP1 with rituximab (HR 1.84, 95%-CI [1.05-3.24], P=0.035). No significant association was observed in GLSG patients treated with CHOP (HR 1.06, 95%-CI [0.73-1.54], P=0.76). High FOXP1 expression was associated with distinct molecular features such as TP53 mutations, expression of IRF4, and gene expression signatures reminiscent of dark zone germinal centre or activated B cells. Conversely, low expression of FOXP1 was associated with - in addition to EZH2 and MEF2B mutations - GNA13 and TNFRSF14 mutations, as well as with expression of genes related to normal germinal centre light zone cells and germinal centre B cells. Taken together, these observations suggest that FOXP1 expression delineates FL cases with distinct genomic and phenotypic characteristics.

Conclusion: In patients treated with immunochemotherapy, high FOXP1 expression identified patient populations with significantly shortened FFS. On the other hand, FOXP1 did not separate distinct outcome groups in patients treated without rituximab, suggesting that FOXP1 is a predictive biomarker. In multivariate analyses, high FOXP1 expression remained significantly associated with FFS in immunochemotherapy patients, after adjusting for the FLIPI. Moreover, FOXP1 high and low expression phenotypes are reflective of distinct sets of molecular alterations. Our results illustrate the value of understanding the intricate relationships between lymphoma biology and response to treatment.