Progression of Disease Within 24 Months in Follicular Lymphoma Is Associated With Reduced Intratumoral Immune Infiltration

Abstract

Purpose: Understanding the immunobiology of the 15% to 30% of patients with follicular lymphoma (FL) who experience progression of disease within 24 months (POD24) remains a priority. Solid tumors with low levels of intratumoral immune infiltration have inferior outcomes. It is unknown whether a similar relationship exists between POD24 in FL.

Patients and methods: Digital gene expression using a custom code set-five immune effector, six immune checkpoint, one macrophage molecules-was applied to a discovery cohort of patients with early- and advanced-stage FL (n = 132). T-cell receptor repertoire analysis, flow cytometry, multispectral immunofluorescence, and next-generation sequencing were performed. The immune infiltration profile was validated in two independent cohorts of patients with advanced-stage FL requiring systemic treatment (n = 138, rituximab plus cyclophosphamide, vincristine, prednisone; n = 45, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone), with the latter selected to permit comparison of patients experiencing a POD24 event with those having no progression at 5 years or more.

Results: Immune molecules showed distinct clustering, characterized by either high or low expression regardless of categorization as an immune effector, immune checkpoint, or macrophage molecule. Low programmed death-ligand 2 (PD-L2) was the most sensitive/specific marker to segregate patients with adverse outcomes; therefore, PD-L2 expression was chosen to distinguish immune infiltration^HI (ie, high PD-L2) FL biopsies from immune infiltration^LO (ie, low PD-L2) tumors. Immune infiltration^HI tissues were highly infiltrated with macrophages and expanded populations of T-cell clones. Of note, the immune infiltration^LO subset of patients with FL was enriched for POD24 events (odds ratio [OR], 4.32; c-statistic, 0.81; P = .001), validated in the independent cohorts (rituximab plus cyclophosphamide, vincristine, prednisone: OR, 2.95; c-statistic, 0.75; P = .011; and rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone: OR, 7.09; c-statistic, 0.88; P = .011). Mutations were equally proportioned across tissues, which indicated that degree of immune infiltration is capturing aspects of FL biology distinct from its mutational profile.

Conclusion: Assessment of immune-infiltration by PD-L2 expression is a promising tool with which to help identify patients who are at risk for POD24.

FIG 1.

Unsupervised hierarchical clustering identifies immune infiltration^HI and immune infiltration^LO follicular lymphoma (FL) tissues. Immune effector, immune checkpoint, and macrophage gene expression in the FL tissue discovery cohort (n = 132) was digitally quantified by NanoString. Green denotes low and red indicates high gene expression. Genes were categorized as follows: immune effector (CD137, CD4, CD7, CD8A, TNFα); immune checkpoint (PD-1, PD-L1, PD-L2, TIM3, LAG3, FOXP3); and macrophage (CD68) molecules. POD, progression of disease; POD24, progression of disease within 24 months.

FIG 2.

Immune infiltration^HI and immune infiltration^LO follicular lymphoma (FL) tissues show distinct gene and protein expression patterns. (A) Fluorescence-activated cell sorting was used to sort cells from deaggregated FL tissues and PD-L2 was quantified by quantitative polymerase chain reaction, normalized to GAPDH and adjusted for the relative proportion of CD20⁺ to non-CD20⁺ cells. Histogram with mean and 95% CIs are shown. The effect size (ie, the measure of the magnitude of the increase in PD-L2 in non-B cells), as conveyed by the median of differences for the samples, is 0.007122. (B) Dichotomizing FL samples as immune infiltration^HI or immune infiltration^LO on the basis of PD-L2 gene expression showed significantly higher gene expression of multiple immune molecules (effector, checkpoint, and macrophages). (C) Stratification of FL tissues by the top and bottom quartiles of PD-L2 expression highlighted the clustering of immune genes. (D) PD-L2 is used to stratify between immune infiltration^HI and immune infiltration^LO tissues in which flow cytometry was performed at diagnosis on fresh deaggregated FL tissues. HK, housekeeping genes; ns, not significant. (*) P = .05 to .01; (†) P = .001 to .0001; (‡) P < .0001.

FIG 3.

(A) Immune-infiltration^HI tumors demonstrate a higher productive clonality of the TCR repertoire in the FL tumor microenvironment. Clonality describes the degree to which one or a few clones dominate the repertoire. (B) Quantitative MIF staining demonstrates increased protein expression of key immune-effectors (CD8) and immune-checkpoints (PD-L1) in immune-infiltration^HI tumors. (C) Quantitative multispectral immunofluorescence (MIF) staining demonstrates increased protein expression of key immune effectors (CD8), immune checkpoints (PD-L1), and macrophages (CD68) in immune infiltration^HI tumors. MIF images demonstrating increased immune infiltrate and intensity of immune effectors, checkpoints, and macrophage molecules in immune infiltration^HI tumors. (D) Quantitatively, PD-L1 expression is upregulated on macrophages in immune infiltration^HI tumors.

FIG 4.

Low immune infiltration cases are enriched in progression of disease within 24 months (POD24) events. The proportion of POD24 events occurring in the (A) Princess Alexandra Hospital (PAH) discovery cohort (B) patients in the PAH discovery cohort with active treatment, advanced stage (C) British Columbia Cancer Agency (BCCA) validation cohort and (D) German Low Grade Lymphoma Study Group 2000 (GLSG2000) validation cohort were stratified by the degree of immune infiltration. OR, odds ratio.

FIG 5.

Distribution of gene mutations in immune infiltration^HI and immune infiltration^LO follicular lymphoma lymph nodes. Relative frequency of FL relevant mutations stratified by immune infiltration^HI and immune infiltration^LO gene expression. No significant differences were seen for any mutation.