A gene-expression profiling score for prediction of outcome in patients with follicular lymphoma: a retrospective training and validation analysis in three international cohorts

Abstract

Background: Patients with follicular lymphoma have heterogeneous outcomes. Predictor models to distinguish, at diagnosis, between patients at high and low risk of progression are needed. The objective of this study was to use gene-expression profiling data to build and validate a predictive model of outcome for patients treated in the rituximab era.

Methods: A training set of fresh-frozen tumour biopsies was prospectively obtained from 160 untreated patients with high-tumour-burden follicular lymphoma enrolled in the phase 3 randomised PRIMA trial, in which rituximab maintenance was evaluated after rituximab plus chemotherapy induction (median follow-up 6·6 years [IQR 6·0-7·0]). RNA of sufficient quality was obtained for 149 of 160 cases, and Affymetrix U133 Plus 2.0 microarrays were used for gene-expression profiling. We did a multivariate Cox regression analysis to identify genes with expression levels associated with progression-free survival independently of maintenance treatment in a subgroup of 134 randomised patients. Expression levels from 95 curated genes were then determined by digital expression profiling (NanoString technology) in 53 formalin-fixed paraffin-embedded samples of the training set to compare the technical reproducibility of expression levels for each gene between technologies. Genes with high correlation (>0·75) were included in an L2-penalised Cox model adjusted on rituximab maintenance to build a predictive score for progression-free survival. The model was validated using NanoString technology to digitally quantify gene expression in 488 formalin-fixed, paraffin-embedded samples from three independent international patient cohorts from the PRIMA trial (n=178; distinct from the training cohort), the University of Iowa/Mayo Clinic Lymphoma SPORE project (n=201), and the Barcelona Hospital Clinic (n=109). All tissue samples consisted of pretreatment diagnostic biopsies and were confirmed as follicular lymphoma grade 1-3a. The patients were all treated with regimens containing rituximab and chemotherapy, possibly followed by either rituximab maintenance or ibritumomab-tiuxetan consolidation. We determined an optimum threshold on the score to predict patients at low risk and high risk of progression. The model, including the multigene score and the threshold, was initially evaluated in the three validation cohorts separately. The sensitivity and specificity of the score for the prediction of the risk of lymphoma progression at 2 years were assessed on the combined validation cohorts.

Findings: In the training cohort, the expression levels of 395 genes were associated with a risk of progression. 23 genes reflecting both B-cell biology and tumour microenvironment with correlation coefficients greater than 0·75 between the two technologies and sample types were retained to build a predictive model that identified a population at an increased risk of progression (p<0·0001). In a multivariate Cox model for progression-free survival adjusted on rituximab maintenance treatment and Follicular Lymphoma International Prognostic Index 1 (FLIPI-1) score, this predictor independently predicted progression (adjusted hazard ratio [aHR] of the high-risk group compared with the low-risk group 3·68, 95% CI 2·19-6·17 [p<0·0001]). The 5-year progression-free survival was 26% (95% CI 16-43) in the high-risk group and 73% (64-83) in the low-risk group. The predictor performances were confirmed in each of the individual validation cohorts (aHR comparing high-risk to low-risk groups 2·57 [95% CI 1·65-4·01] in cohort 1; 2·12 [1·32-3·39] in cohort 2; and 2·11 [1·01-4·41] in cohort 3). In the combined validation cohort, the median progression-free survival was 3·1 years (95% CI 2·4-4·8) in the high-risk group and 10·8 years (10·1-not reached) in the low-risk group (p<0·0001). The risk of lymphoma progression at 2 years was 38% (95% CI 29-46) in the high-risk group and 19% (15-24) in the low-risk group. In a multivariate analysis, the score predicted progression-free survival independently of anti-CD20 maintenance treatment and of the FLIPI score (aHR for the combined cohort 2·30, 95% CI 1·72-3·07).

Interpretation: We developed and validated a robust 23-gene expression-based predictor of progression-free survival that is applicable to routinely available formalin-fixed, paraffin-embedded tumour biopsies from patients with follicular lymphoma at time of diagnosis. Applying this score could allow individualised therapy for patients according to their risk category.

Funding: Roche, SIRIC Lyric, LYSARC, National Institutes of Health, the Henry J Predolin Foundation, and the Spanish Plan Nacional de Investigacion.

Figure 1. Outline of the overall study design

Fresh-frozen tissue (FFT) tumor biopsies were prospectively obtained from 160 untreated patients enrolled in the international PRIMA trial. RNA with sufficient quality (RIN>6.5) was obtained for 149/160 cases and gene-expression profiling was performed using Affymetrix U133 Plus 2.0 micro-arrays. A multivariate Cox regression analysis identified genes whose expression was associated with PFS independent of maintenance treatment in the subgroup of randomized patients. Expression levels from 95 curated genes were then determined by means of digital expression profiling (NanoString technology) in 53 FFPET samples of the training set, allowing assessment of the technical replication of expression levels for each gene between technologies. Genes with high correlation (>0.75) were included in a L2-penalized Cox model adjusted on rituximab maintenance to build a PFS-predictive score. The model was further evaluated using NanoString technology in 488 FFPET samples from 3 independent international cohorts of patients: a distinct validation set from the PRIMA trial (n=178), and two others obtained in large centers (respectively the Mayo Clinic/Iowa SPORE project, n=201 and the Barcelona Hospital Clinic, n=109). An unsupervised analysis of the gene-expression data generated in the training cohort was also performed independently. Abbreviations: FFT: fresh-frozen tissues; FFPE: formalin-fixed paraffin-embedded tissues; PFS: progression-free survival.

Figure 2. Progression-free survival of patients from the training cohort, according to the predictor score

Kaplan-Meier estimates of progression-free survival in randomized patients of the training cohort, since the time of randomization in PRIMA trial. An optimal threshold was set to separate patients into high- (n=47, 35% of the patients, red curve) and low-risk (n=87, 65% of the patients, blue curve) groups with significantly different outcomes (p<0.0001, log rank test). The 5-year PFS rates were 26% (95%CI: 16-43) and 73% (95%CI: 64-83) for the high-and low-risk groups, respectively. For each time point the number of patients at risk and (number of patients censored) are indicated. Abbreviations: PFS: progression-free survival.

Figure 3. The gene expression-based predictor for FL patients tested in the validation cohorts

The predictor is a linear combination of the log₂-transformed normalized gene expression levels weighted by individual gene coefficients. A: The relative gene expression levels of the 23 genes in the predictive model are presented in the form of a heat map. Each column represents a single patient from the combined validation cohorts, arranged according to the predictor score, with lowest score on the left. Each row represents a gene from the model, ordered by gene contribution to the score. B: The score from the predictor for patients in the validation cohorts. The patients are arranged as in panel A. The vertical red line separates patients into high- (n=122, 27% of the patients) and low-risk (n=338, 73% of the patients) groups according to the threshold (horizontal line) determined in the training cohort. The clinical and treatment characteristics of the patients are depicted. Cohort 1 included patients from the PRIMA trial, cohort 2 from the University of Iowa/Mayo Clinic Lymphoma SPORE and cohort 3 from the Hospital Clinic University of Barcelona. C: The relative contributions of each of the 23 genes to score variation. The X axis position of the boxes represents the absolute average contribution of the genes (calculated as the mean expression in a given cohort, multiplied by the coefficient assigned to the gene in the score). The width of the boxes shows the contribution of each gene to the score variation (calculated as the standard deviation of the gene in the cohort multiplied by its coefficient). Gene contributions are presented in both the training cohort (grey) and the combined validation cohort (black).

Figure 4. Kaplan-Meier estimates of progression-free survival predicted by the 23-gene signature score among patients of the three validation cohorts and according to FLIPI score

The threshold set in the training cohort separated patients into high- and low-risk groups (red and blue curves, respectively). The 23-gene score significantly predicted PFS in patients from each validation cohort (A-C: Cohort 1, 2, and 3, respectively) as well as in the combined validation cohorts (D) and in each FLIPI subgroup (E-G: low, intermediate and high risk FLIPI scores, respectively). Logrank test p-values for each of the comparisons are reported. FLIPI score was available for 453 patients. Abbreviations: PFS: progression-free survival; 95%CI: 95% confidence interval.