Cessation of Somatic Hypermutation of Immunoglobulin Genes in Follicular Lymphomas after Isotype Switching Despite Continuous Expression of Activation-Induced Cytidine Deaminase (AID).
Authors: Bertinetti Cristina , Weiss Julia , Veelken Hendrik 
Published: October 2019

Abstract
Follicular Lymphomas (FL) are considered the neoplastic counterpart of germinal center (GC) B cells due to their characteristic growth pattern, composition of a mixture of centroblasts and centrocytes, a GC gene expression profile, and ongoing acquisition of somatic mutations of their immunoglobulin (Ig) genes. A minority of FL has undergone Ig class switching from IgM to IgG or IgA at diagnosis. Based on PCR-mediated cloning of Ig heavy chain (IgH) genes with a success rate of 73%, Aarts et al. (Blood, 2000) have reported from an analysis of 30 FL cases that isotype-switched FL harbor significantly more IgH mutations than IgM-expressing cases, suggesting that the somatic hypermutation (SHM) machinery remains fully active even after class switch recombination (CSR) in FL. We have readdressed this question in 38 FL cases and compared their SHM pattern to 31 lymphomas of other types. 16 of these controls expressed IgM (including 5 MCL, 4 DLCL, and 3 CLL) and 15 IgG/A (including 13 myelomas). The Ig isotype and light chain (IgL) restriction of the lymphoma cells were identified by flow cytometry, and both clonal IgH and IgL transcripts were cloned by anchored PCR. This approach has a success rate of 98% (Bertinetti et al., Eur. J. Haematol., 2006). At the DNA (Table 1 and 2) and amino acid sequence level (not shown), the SHM frequency did not differ significantly between the 23 IgM-expressing and 15 class-switched FL. IgM-expressing FL, however, had more SHM than unswitched non-FL cases. In contrast, class-switched FL did not harbor more mutations than switched non-FL tumors. Although the molecular mechanisms are not yet fully understood, activation-induced cytidine deaminase (AID) has been identifed as an essential enzyme required for both SHM and CSR and is frequently expressed by GC lymphomas. To investigate whether downregulation of AID might be responsible for cessation of SHM after CSR in FL, we measured the AID expression in the FL biopsies by a semiquantitative RT-PCR. In contrast to our hypothesis, AID transcripts were present in both IgM- and IgG/A-expressing FL, and the median AID expression was even significantly higher in switched FL (p=0.014). These data indicate that despite their arrest in the GC maturation stage, FL behave similar to normal B cells by downregulating the SHM machinery after CSR. In addition, the data are consistent with a model in which intermediate expression of AID is sufficient for SHM, but higher levels are required to initiate CSR in FL. Subsequent cessation of SHM might be attributable to subcellular relocalisation of AID or decreased activity of AID cofactors, such as single-stranded binding protein (RPA) or protein kinase A (PKA).
Median (range) of VH Mutations IgM IgG/A IgM versus IgG/A Comparison of mutation frequency of the clonal VH gene between non-switched and switched FL cases and between FL and non-FL cases FL (n=38) 32 (8–106) 38 (12–58) p=0.4 Non-FL (n=31) 18 (6–57) 25 (13–65) p=0.07 FL versus non-FL p=0.006 p=0.11 Median (range) of VL Mutations IgM IgG/A IgM versus IgG/A Comparison of mutation frequency of the clonal VL gene between non-switched and switched FL cases and between FL and non-FL cases FL (n=38) 14 (0–38) 18 (5–31) p=0.36 Non-FL (n=31) 5.5 (0–25) 14 (5–53) p=0.001 FL versus non-FL p=0.002 p=0.32