Very low-dose irradiation (2 ؋ 2 Gy) is a new, effective, and safe local treatment for follicular lymphoma. To understand the biologic mechanisms of this extremely effective response, we compared by microarray the gene-expression profile of patients' biopsies taken before and after radiation. In all patients, a major and consistent induction of p53 target genes was seen. p53 targets involved in cellcycle arrest and apoptosis showed the same mode of regulation, indicating that, in vivo, both are activated simultaneously. p53 up-regulation and p53-mediated proliferation arrest and apoptosis were substantiated using immunohistochemistry, with activation of both the intrinsic and the extrinsic apoptotic pathways. The other induced genes revealed a whole set of biologically meaningful genes related to macrophage activation and TH1 immune response. Immunohistochemical analysis suggested a specific activation or differentiation of resident macrophages by apoptotic cells. These biologic insights are important arguments to advocate the use of low-dose radiotherapy as an effective palliative treatment for follicular lymphoma. Moreover, this study is the first in vivo report of the radiationinduced p53 apoptotic response in patients and suggests that this apoptotic response is not immunologically silent.

Involved field radiation therapy with 30 to 40 Gy is a valuable local treatment for follicular lymphoma (FL) that is routinely used in clinical practice. We previously showed that very low dose radiation (2x2 Gy, days 1 and 3) is also effective, with rapid and often long lasting remissions in up to 90% of FL patients (Haas et al, JCO, 2003). However, the biological mechanism of this extremely effective response is not known. To study the molecular response to low dose radiation therapy in FL, gene-expression profiling using 35K spotted 60-mer oligo-arrays was performed from lymph node biopsy samples taken before treatment and 24 hours after the second dose of 2 Gy irradiation, in 15 patients. The clinical response was excellent (10 CR, 5 PR). In all patients, a major and consistent induction of p53 and p53 target genes was seen, reflecting both proliferation arrest (e.g., P21, repression of cell-cycle regulated genes) and apoptosis induction (NOXA, PUMA, BAX, TRAIL/DR5 and FAS). P53 upregulation, p53-mediated proliferation arrest and apoptosis were substantiated using immunohistochemistry with dramatic increase of p53 protein levels in B-cells, T-cells and accessory cells. There was also a significant increase in the numbers of cleaved-caspase 8 positive cells (death receptor pathway apoptosis) with a minor increase of cleaved-caspase 3 positive cells and morphological features of apoptosis, suggesting a relatively early stage in the apoptotic process. The other induced genes revealed an ‘immune signature’, with a whole set of biologically meaningful genes related to macrophages (e.g., CD68, TLR4), TH1 immune response (e.g., IL18, CXCL9, 10, 11), clearance of apoptotic cells (e.g., C1Q, lysosomal enzymes), tolerance (ILT-3, IL-4, IDO) and death receptor ligands (FASL, TRAIL). Immunohistochemical analysis did not show an increase in T-cell subsets and macrophages density, rather suggesting an activation or differentiation of resident macrophages by radiation and/or apoptotic cells than recruitment of novel cell populations. This is the first global analysis of the direct molecular effect of radiotherapy and p53 related apoptosis in vivo in human lymphoma. Moreover, the ‘immune signature’ suggests that radiation-induced apoptosis in FL is not an immunologically silent process, but rather an early event that could contribute to the death and clearance of tumor cells. These insights may have important implications for modulation of the cancer-related immune response and for immunotherapeutical approaches in FL.