Role of activation-induced cytidine deaminase (AID) in follicular lymphoma biology

Follicular lymphoma is the second most common non-Hodgkin’s lymphoma (NHL). The clinical course of disease is heterogeneous, typically with multiple relapses. Most patients live 10 years or more. However, another group of patients deteriorate rapidly and may progress to death within two years. Activation induced cytidine deaminase (AID) is an enzyme that plays an important role in somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin genes (IG). It induces mutations in IG and non-IG genes leading to genomic instability and chromosomal breaks that are important in the pathogenesis of B-cell malignancies. In this study, we wanted to first measure AID mRNA and protein levels and its biological function in follicular lymphoma (FL) and then correlate each of these variables with clinical features. Our cohort consisted of 87 patients recruited into the Purine-Alkylator Combination In Follicular lymphoma Immuno-Chemotherapy for Older patients (PACIFICO) trial which is comparing alternative frontline chemoimmunotherapy regimens in older patients with FL. The patient samples were in the form of formalin fixed, paraffin embedded (FFPE) biopsies, which are notorious for nucleic acid degradation. We first chose the best kits for extracting RNA and DNA from FFPE biopsies then optimized the procedure to obtain higher quantity of RNA and DNA from the minimum amount of tissue. We then degraded RNA from an AID positive cell line by heating and compared the degraded material with intact material obtained from the same cells to identify a cut-off point for RNA degradation to be applied in a quantitative polymerase chain reaction (qPCR) experiments. This was followed by a qPCR experiment to identify AID mRNA expression in 59 patients. AID protein was then quantified by Immunohistochemistry (IHC) in all samples. We also aimed to measure the functional readout of AID, first by exploring the nuclear/cytoplasmic (N/C) ratio of AID 2 (AID is stored in the cytoplasm and translocates to the nucleus to function), which was calculated for 20 patients using confocal microscopy. A second AID functional measurement was applied using cloning and PCR to detect ongoing mutation and AID-induced mutation in the immunoglobulin heavy variable gene (IGHV) in 18 cases. Finally, we correlated AID expression and functional readouts with available baseline and longitudinal clinical data obtained from the Clinical Trials Unit. In summary, a significant positive correlation was found between AID mRNA and protein expression (P= 0.001). We also found a significantly higher AID N/C ratio in the patient group with higher total AID mRNA and protein expression (P= 0.025 and 0.023 respectively). No correlation was identified between AID mRNA or protein levels and baseline or longitudinal clinical data. However, AID functionality measured as N/C ratio of AID and AID-related or ongoing IGHV mutation was positively correlated with disease status, treatment response and patient survival times. In conclusion, we found that functional readouts of AID are more strongly associated with adverse clinical features in FL compared to AID mRNA or protein expression.