Background: The use of epigenetic modifier drugs such as EZH2 inhibitors has recently expanded. However, since the clinical course of follicular lymphoma (FL) is heterogenous, there are still challenges in identifying patients who will benefit from EZH2 inhibitors in general hospital settings without next-generation sequencing.
Aims: We aimed to validate the epigenetic characteristics of EZH2 expression and H3K27 methylation status using immunohistochemistry staining. We also examined the usefulness of FLIPI, POD24, and cell-free DNA concentration in a recent cohort.
Methods: Altogether, 164 patients (female, n=91; male, n=73) diagnosed with FL at Kyoto University Hospital between 2005 and 2020 were included. Immunohistochemical (IHC) EZH2 expression and H3K27 methylation profiles were evaluated in 105 patients. EZH2 expression was assessed using the H-score (positivity rate (%) x staining intensity: negative, 0; weak, 1; moderate, 2; strong, 3), and H3K27 methylation profile was assessed by two independent estimators using the H3K27me3/me2 IHC scoring system, based on a previous report. Cell-free DNA concentration was examined at diagnosis or prior to the first chemotherapy in a total of 18 patients, out of whom 12 patients were assessed sequentially at 1, 3, 6, and 12 months after the first examination.
Results: The median age at diagnosis was 61 years (range, 33-86) and the median follow-up period was 7.5 years. FLIPI at diagnosis was high in 46 patients (28.3%), intermediate in 60 patients (37.0%), and low in 47 patients (29.0%). A total of 90 patients received rituximab (RTX)-containing chemotherapy (BR, n=10; R-CHOP/R-CVP, n=68; RTX monotherapy, n=9; other regimen, n=3), 62 patients were observed without any intervention (watchful waiting), and 5 patients underwent resection of the tumors. Of those with watchful waiting, 27 experienced lymphoma progression and received chemotherapy. H-scores of EZH2 ranged from 0 to 300 (mean score, 92) and H3K27me3/me2 score ranged from -4.2 to 5.0 (mean score, 2.3). A high H3K27me3/me2 score (≥ 2) was associated with superior OS, and the combination of low H3K27me3/me2 score (<2) and high EZH2 score (≥ 100) was associated with inferior OS (p=0.009) with high frequency rapid progression (62.5%) within 2 years after the 1st immunochemotherapies (Figure 1A, B). Cell-free DNA concentration ranged from 0.45 ng/µl to 85.60 ng/µl (median conc, 0.67 ng/µl), which showed no apparent correlation with the serum level of LDH, sIL-2R, or imaging status. POD24 was detected in 38 patients, and its impact on OS was not statistically significant (HR 8.1, p=0.070). No patients with FLIPI-Low died during the follow-up, and only FLIPI-Int was associated with inferior OS (6/60 died), but not FLIPI-High (1/46 died).
Image:
Summary/Conclusion: Our study suggested that H3K27me3/me2 and EZH2 scores of the tumor samples at diagnosis could predict their outcomes after immunochemotherapy. As high H3K27me3/me2 is known to be associated with EZH2 mutations, good prognosis of those with high H3K27me3/me2 scores might reflect the superior impact of EZH2 mutations, as reported in m7FLIPI. Meanwhile, tumors presenting low H3K27me3/me2 scores with high EZH2 scores have the potential to rely on other driver mutations for pathogenicity, although genetic analysis remains under investigation. FLIPI and POD24 were not good predictors of survival in our cohort, which might be due to our regimen choice and the frequency of imaging follow-up. More experiments are needed to apply cell-free DNA for clinical use in patients with FL.