Neither CD68+ Nor CD163+ Macrophages Are Associated with Decreased Survival in Follicular Lymphoma
Authors: Gratzinger Di ta , Ai Weiyun , Tibshirani Robert , Levy R. , Natkunam Y. 
Published: October 2019

Abstract
Introduction: Tumor-infiltrating immune cells are thought to be important in the biology and clinical course of follicular lymphoma. Recently a gene expression signature associated with monocyte/macrophages was identified in follicular lymphoma patients with poor overall survival. One hypothesis is that macrophages may act to suppress the immune response to the lymphoma and thus decrease survival. A number of groups have assessed the prognostic value of enumerating tumor-associated macrophages (TAM) using immunohistochemistry for the macrophage marker CD68, with mixed results: some have shown poorer survival with increased CD68+ TAM, others have shown no effect, and others have shown poorer survival only in patients not treated with rituximab. CD68 is a sensitive marker of tissue macrophages which also shows reactivity with other cell types. CD163, by contrast, shows high specificity for the monocyte/macrophage lineage and is thought to be a marker for immunosuppressive M2-polarized macrophages.
Methods: 187 follicular lymphoma patients underwent excisional lymph node biopsy prior to treatment. We enumerated extrafollicular and follicular CD163+ and CD68+ TAM in two high power fields (hpf) each of duplicate tissue microarray cores. The median age at diagnosis was 45 yrs, with a median follow-up of 8.8 yrs and 51 deaths. All but 6 patients had stage III/IV disease, 3 had stage I/II, and 3 were unknown. The histological grades are as follows: 112 grade 1, 65 grade 2, and 10 grade 3. FLIPI (follicular-lymphoma specific international prognostic index) scores are as follows: 33 low, 95 intermediate, 19 high, 40 unavailable. For their initial treatment regimen 120 patients received CVP +/− fludarabine; 42 received a variety of other chemotherapeutic regimens; and 9 patients received rituximab.
Results: CD163 marks a subset of TAMs that is preferentially enriched in the extrafollicular compartment and that does not covary with numbers of CD68+ TAMs overall. CD163+ TAMs are significantly less frequent than CD68+ TAMs: 32 vs 88 TAM/hpf in the extrafollicular compartment (p = 5.89E-62) and 2.5 vs 29 TAM/hpf in the follicular compartment (p = 2.05E-62). Both CD163+ and CD68+ TAMs are enriched in the extrafollicular compartment (p = 5.39E-46 for CD163, p = 2.17E-81 for CD68); however this gradient is more marked for CD163+ than for CD68+ TAMs (7.6% of CD163+ TAM vs 24% of CD68+ TAM are follicular, p = 6.53E-43). There is no correlation between numbers of CD68+ and CD163+ TAM in the follicular (r2 = 0.05) or extrafollicular (r2 = 0.12) compartments. Furthermore, while CD68+ TAM content within follicles increases with grade (26/hpf in grade 1 vs 33/hpf in grade 2 follicular lymphoma, p = 0.01) there is no corresponding increase in CD163+ TAM within follicles (2.8/hpf in grade 1 vs 2.0 per hpf in grade 2 FL, p = 0.15). There was no association between TAM content and FLIPI. We found no statistical difference in overall survival with respect to either CD163+ or CD68+ TAM within follicles or in the extrafollicular compartment. Higher numbers of extrafollicular CD68+ macrophages are associated with longer initial treatment-free interval (time from diagnosis to initial treatment; z = −2.23, p = 0.026). This association is independent of FLIPI and grade (z = −2.94, p =0.0033). Higher numbers of extrafollicular CD163+ macrophages are associated with a longer second treatment-free interval (interval from first treatment to second treatment; z = −2.151, p = 0.031); this association is independent of FLIPI and grade (z = −2.151, p = 0.031).
Conclusions: It has been suggested that CD68+ TAM are associated with poorer prognosis in follicular lymphoma; however we found no association with overall survival, and indeed a longer initial treatment-free interval, with increased CD68+ extrafollicular macrophages. CD163 is highly expressed in immunosuppressive M2-polarized macrophages, which are thought to provide an environment permissive to cancer progression. We found that CD163+ macrophages are a small subset of the total CD68+ macrophage count, are distributed differentially, and vary independently of CD68+ macrophage content, suggesting that CD163 highlights a functionally distinct macrophage subpopulation. However CD163+ macrophages, like CD68+ macrophages, were not associated with poorer overall survival and indeed were associated with a longer second treatment-free interval (time from first to second treatment).