Adjuvant rituximab and elevated intratumoural CD8 expression are associated with sustained disease control after radiotherapy in a randomised trial of systemic therapy in early-stage follicular lymphoma

Abstract

Background: We report extended follow-up of TROG99.03, a randomised phase III trial in early-stage follicular lymphoma (ESFL) including new information on the role of adjuvant rituximab and translational studies.

Methods: Patients with ESFL were randomised to involved field radiotherapy (IFRT) or IFRT plus 6-cycles cyclophosphamide/vincristine/prednisolone (IFRT + CVP). From 2006 rituximab was added to IFRT + CVP (IFRT + R-CVP). Clinical and multi-omic parameters were evaluated. Findings were validated in two independent ESFL cohorts (99 and 60 patients respectively).

Findings: Between 2000 and 2012, 150 (75 per arm) patients were recruited. 48% were positron emission tomography (PET)-staged. By protocol, at median follow-up 11.3-years, progression-free survival (PFS) remained superior for IFRT+(R)CVP vs. IFRT (hazard ratio [HR] = 0.60, 95% CI = 0.37-0.98, p = 0.043; 10-year PFS 62% vs. 43%) respectively. Although no significant difference in overall survival was observed (HR = 0.44, 95% CI = 0.16-1.18, p = 0.11, 10-year OS 95% vs. 84%), patients receiving IFRT+(R)CVP experienced fewer composite (histological transformation and death) events (p = 0.045). PFS of IFRT + R-CVP-treated patients compared with all other treatments lacking rituximab (IFRT alone plus IFRT + CVP) was superior (HR = 0.36, 95% CI = 0.13-0.82, p = 0.013). Amongst PET-staged patients, PFS differences between IFRT + R-CVP vs. IFRT were maintained (HR = 0.38, 95% CI = 0.16-0.89, p = 0.027) indicating benefit distinct from stage migration. FL-related mutations and BCL2-translocations were not associated with PFS. However, by multivariate analysis elevated CD8A gene expression in diagnostic biopsy tissue was independently associated with improved PFS (HR = 0.45, 95% CI = 0.26-0.79, p = 0.037), a finding confirmed in both ESFL validation cohorts. CD8A gene expression was raised (p = 0.02) and CD8+ T-cell density higher within follicles in ESFL vs. advanced-stage FL (p = 0.047). Human leucocyte antigen class I specific neoantigens were detected in 43% of patients, suggesting neoantigen-specific CD8+ T-cells have a role in confining the spread of the disease.

Interpretation: Adjuvant R-CVP and elevated intratumoural CD8 expression were independently associated with sustained disease control after radiotherapy in ESFL.

Funding: Cancer Council Victora; National Health and Medical Research Council; Leukaemia Foundation; Mater Foundation.

Keywords: CD8; Early-stage follicular lymphoma; Neoantigen. randomized clinical trial; Radiotherapy; Rituximab.

Fig. 1

Outcomes of 150 patients with ESFL registered to the TROG99.03 trial with extended follow-up. A) Clinical trial schema outlining randomization and treatment received (TR) to IFRT vs. combined modality treatment (CMT) (i.e. IFRT+(R)CVP) arms. B) Progression-free survival of IFRT vs. IFRT+(R)CVP arms. C) Overall survival of IFRT+(R)CVP vs. IFRT arms. D) Hazard curves demonstrating cumulative incidence of death and histologic transformation in IFRT+(R)CVP and IFRT treatment arms.

Fig. 2

Outcomes based on baseline FDG-PET staging, period, and rituximab. Progression-free survival by ITT analysis of IFRT+(R)CVP vs. IFRT arms in the A) late-era and B) early era. C) Progression-free survival in all patients in the TROG99.03 trial based on whether baseline FDG-PET was used as a staging modality and D) FDG-PET staged patients by IIT analysis of IFRT+(R)CVP vs. IFRT arms. E) Progression-free survival, per treatment received, of R–CVP treated patients compared with all non-rituximab containing treatments. F) Progression-free survival, per treatment received, of R–CVP treated patients compared with CVP treated patients.

Fig. 3

Waterfall plot demonstrating mutation profile of 101 patients with ESFL enrolled in the TROG99.03 trial. The frequencies and nature of the mutations of the top 12 genes are shown, with patients stratified by BCL2-translocation.

Fig. 4

Progression-free survival stratified by CD8A gene expression in the discovery and validation cohorts, and CD8A/rituximab interaction analysis. A) TROG99.03 discovery cohort. B) AusESFL validation cohort. C) CanESFL validation cohort. D) TROG99. 03 discovery cohort stratified by rituximab use and CD8A gene expression.

Fig. 5

T-cell Enrichment in the ESFL tumour microenvironment. A) Digital gene expression of T-cell genes is increased in ESFL compared with ASFL. B) The CD8 intrafollicular:extrafollicular (IF:EF) ratio as measured by immunohistochemistry demonstrates significant enrichment by of CD8+ T-cells in interfollicular regions. C) Representative cases of low IF:EF ratio from diagnostic biopsy of an ASFL patient (left) and high IF:EF ratio from diagnostic biopsy of an ESFL (right) by light microscopy at 10× and 40× magnification. ∗ denotes p = 0.05–0.01, ∗∗ denotes p < 0.01.

Fig. 6

HLA-class I specific neoantigens in ESFL. Frequency of mutations in the TROG99.03 cases that had available germ-line DNA for HLA-class I genotyping, including ‘disruptive’ (i.e. nonsense and frameshift) and non-disruptive (predominantly missense) mutations. A histogram showing all genes with ≥2 neoantigens is shown in the right panel.