Phd in IINNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Thesis title: EZH2 GENE MUTATIONS AND COPY NUMBER VARIATION AS PROGNOSTIC MARKERS IN ADVANCED FOLLICULAR LYMPHOMA

Introduction Gain-of-function mutations in the Enhancer of Zeste Homolog 2 (EZH2) gene are prevalent in follicular lymphoma (FL) and encode for a histone methyltransferase, which can also be over-expressed by copy number variations (CNV). The clinical impact of EZH2 aberrations inFL is debated, with some studies suggesting a favorable outcome, especially when patients received R-CHOP/CVP. Digital droplet PCR is a promising technique to screen EZH2 aberrations. • Aim To study the feasibility of ddPCR based screening of EZH2 aberrations and explore the incidence and the role of EZH2 in modifying the first line treatment effect in the prospective trial “FOLL12” of the Fondazione Italiana Linfomi (FIL, EudraCT 2012-003170-60), enrolling FL treated either with R-CHOP or bendamustine-rituximab (BR), according to physician’s choice. • Results and methods Leftover DNA samples from bone marrow (BM) aspirates at baseline, centralized at the FIL MRD Network labs, were tested by a multiplex droplet digital PCR (ddPCR) for the detection of EZH2 point- mutations (Y646X, A682V A692G and by a ddPCR CNV assay for the EZH2 region (chr 7q36.1). Mutations and CNV detected were related to clinical features and patients’ outcome by revising FIL FOLL12 trial elcetronic CRFs. Overall, 409 samples were tested for mutations and 359 for CNV. An EZH2 mutation was found in 137 cases (33%), being more frequent in patients with a documented histological infiltration at the BM biopsy (BM+ n=124/309, 40%) than in BM- (n=13/100, 13%). 68 cases were mutated in Y646 (50%), 41 in A682G (30%) and 73 in A692V (53%), with a median variant allele frequency (VAF) of 0.13% (0.01-32%): 102 cases (74%) showed one single mutation, 30 cases two and 6 cases all the 3 mutations. Interestingly, EZH2 mutated patients showed less favorable clinical features than the WT: higher B2M (>ULN 72% vs 53%, p<0.001), lower Hb (Hb<12, 26% vs 15%, p=0.015), more frequent BM+ (91% vs 68%, p<0.001), higher FLIPI-2 (61% HR vs 40%, p<0.001), higher metabolic tumor volume (MTV>200 ml: 71% vs 57%, p=0.011) and maximum tumor dissemination (Dmax>400 mm: 34% vs 21%, p=0.013).Nonetheless, this did not translate into worse outcome (5-year PFS 59% vs 64%, p=0.324). Overall, no difference in PFS was observed between R-CHOP (n=239) and BR-treated (n=170) patients (5-y PFS 60% vs 67%,p=0.652); EZH2 mutations had no statistically significant impact in modifying the treatment effect of either schedule, despite a trend towards better performance of BR vs R-CHOP in EZH2 mutated cases: PFS HR of R-CHOP vs BR was 1.77 (95CI 0.96-3.29, p=0.067) for mutated and 0.89 (95CI0.60-1.32, p=0.564) for WT patients, Figure 4. In a subgroup analysis, the 30 EZH2 mutated patients who received R-CHOP not followed by R maintenance had the worst outcome (5-y PFS 24%, p=0.007). Regarding EZH2 CNV, a gain was found in 50 cases (14%) and a loss in 9 (3%): among the 337 patients analysed for both mutations and CNV, 13 carried both EZH2 mutation and gain (4%), 106 had mutations only (31%) and 31 had gain only (9%). The 50 patients with CN gain had less BM involvement (62% vs 79%, p=0.018) but no differences in other baseline clinical features nor in outcome; interestingly, among R-CHOP treated patients (n=199) those with EZH2 gain only (n=20) showed a trend towards better outcome (5-y PFS: 75% vs 59%, p=0.053) • Conclusions EZH2 aberrations were tested by ddPCR in 409 BM samples from the largest, prospective series of FL, so far: unexpectedly, high prevalence of A692V mutation was found and described trend towards discordant impact of EZH2 mutations vs gains in modifying the effect of first line therapy, so further investigation is needed before using this biomarker for treatment choice modulation. To better address this issue, testing of diagnostic lymph node samples is ongoing.