Mantle cell lymphoma with t(11;14) and unmutated or mutated V_H genes expresses AID and undergoes isotype switch events

Mantle cell lymphoma (MCL) is characterized by a nodal infiltrate of neoplastic cells with a distinct histology and surface immunoglobulin M (sIgM)(D)⁺⁺CD5/19/20/79b⁺CD23^- immunophenotype.¹  At presentation, disease may involve the spleen, bone marrow, and extranodal sites, with circulating abnormal lymphoma cells. A signature t(11;14) abnormality in the IgH locus readily identifies MCL.¹  Molecular mapping indicates ontogeny of the translocation early in B cells at the stage of D_H-J_H (diversity region of the heavy chain-joining region of the heavy chain) recombination.²  Translocation alone is considered insufficient for neoplastic transformation,³  which may occur subsequently via 2 pathways. One subset of MCL derives from a cell of origin with unmutated (UM) V_H genes and the other minor subset derives from a cell with mutated (MUT) genes.^4,5,6 

Somatic mutation of V genes generally localizes to germinal centers (GCs) in secondary follicles.⁷  Isotype switch events mainly follow onset of hypermutation in normal B cells and for both mechanisms, the expression of activation-induced cytidine deaminase (AID) is essential.^8,9  Isotype class switch recombination (CSR) by a deletional mechanism involves double-strand DNA breaks.⁹  Transcription of excised switched circular DNA generates circle transcripts (CTs), a hallmark of active CSR.^9,10  AID deficiency blocks CSR completely.⁹  Isotype switch may occur by additional mechanisms.^11,12  Expression of AID in vivo normally requires CD40 ligand (CD40L) signals.¹³  Circulating normal B cells show no evidence for CSR, although they have a potential to switch with appropriate signals.^14,15  In contrast, circulating chronic lymphocytic leukemia (CLL) B cells appear to have engaged such signals and constitutively express clonally related switched transcripts, AID and CTs.¹⁵  In addition to a possible role in switch, AID expression in CLL associates with mutations in the preswitch Cμ region but not V_H,¹⁶  suggesting a potential to target non-V gene loci.

In this study, we evaluated the occurrence of isotype switch and AID expression in subsets of MCL cells differing by V_H mutational status.

Eighteen MCL cases with blood lymphocytosis, part of a larger study with confirmed diagnosis of MCL,⁵  were evaluated (17 of 18 peripheral blood mononuclear cell [PBMNC] samples; case 11, bone marrow [BM]-derived MNCs). In each, the t(11;14) translocation was verified by karyotype (n = 3) or interphase fluorescence in situ hybridization (FISH; n = 15). Tumor load (% nuclei with CCND1-IgH fusion) was greater than 90% in 8 cases and 43% to 88% in the remainder. Immunophenotype showed sIgM(D)⁺⁺CD5/19/20/79bFMC7⁺CD10^-CD23^-/weak expression. Variant sIg expression was assessed by 3-color flow cytometry with allophycocyanin (APC)-conjugated anti-CD19; phycoerythrin (PE)-conjugated (f(ab′)2) anti-κ or anti-λ light chain; and fluorescein isothiocyanate (FITC)-conjugated (f(ab′)2) anti-μ, anti-α, or anti-γ heavy chain antibodies (Beckman-Coulter, Hialeah, FL; Dakopatts, Glostrup, Denmark).

PBMNCs (> 5 × 10⁶ ) were extracted for RNA and cDNA synthesized using oligo-dT.¹⁷  Tumor-derived V_H genes were identified using replicate polymerase chain reaction (PCR) and cloning procedures.^5,17  Eight to 12 tumor-derived clones were fully sequenced. Homology to germ line V_H of 98% or above denoted UM status. For identification of variant tumor-derived isotype switch transcripts, specific CDR3-based primers were used in a nested reverse transcriptase-PCR (RT-PCR) protocol.¹⁷  I_H- and C_H-exon-specific primers were used to assay CTs as described.¹⁵  CT-amplified products of predicted size were directly sequenced.¹⁵ 

AID expression was analyzed by standard RT-PCR: forward, 5′-ATGGACAGCCTCTTGATGAAC; reverse, 5′-CTCGTAAGTCATCAACCTCATACA.¹⁸  The primers distinguish wild-type and splice variants of AID on the basis of size. Positivity was assessed after 30 cycles. PBMNCs from 4 of 4 healthy cases were also analyzed.

Of the MCL cases, 11 of 18 were UM and 7 of 18 MUT, displaying intraclonal homogeneity, with cohort mutational status reported previously.⁵ 

In 4 of 6 UM cases and in 4 of 7 MUT cases, variant tumor-derived isotype switch transcripts were detected using specific CDR3-based primers (Figure 1). Cα,γ transcripts were observed in both subsets, with Cϵ in 1 MUT case (Figure 1 and Table 1). Identification of mature VDJ-Cα,γ,ϵ variant transcripts indicates activation of switch mechanisms in vivo.

AID mRNA expression is restricted to GC centroblasts or centrocytes in normal B cells.^15,19,20  Recently, 3 UM cases of MCL and 4 cases of MCL undefined by V_H mutation were separately reported as negative for AID transcripts.^20,21  Interestingly, we observed that in both UM and MUT MCL cells, AID expression is a common feature. The primary wild-type (wt) transcript for AID was identified in 9 of 11 UM and 7 of 7 MUT cases (Figure 2 and Table 1). A splice variant transcript lacking exon 4 was identified,^20,21  predominantly in UM MCLs (10 of 11 cases; Figure 2 and Table 1), confirmed in each case by sequence analysis (data not shown). No other AID variant transcripts were observed.^19,20 AID expression is most likely to be tumor cell related, since normal circulating B cells are devoid of AID expression under assay conditions employed here,^15,19,20  as confirmed in 4 of 4 controls (data not shown). Somatic mutation is not ongoing in MUT MCL cells, and AID appears dissociated from V_H gene mutational activity. This differs from findings in other non-Hodgkin lymphomas, where AID expression assayed by RT-PCR always correlates with somatically mutated cells²⁰  or with a GC phenotype.²¹  It also differs from CLL where AID expression associates largely with unmutated V_H.¹⁹ 

AID expression accompanied VDJ-C_H switch events in 8 of 8 MCL cases but was observed in 3 cases in the absence of such transcripts (Figure 2 and Table 1), indicating dissociation from switch activity when assessing the whole-tumor population. In those cases displaying variant transcripts, we sought evidence for CSR events by examining CT expression.^10,15  In 5 of 7 cases where material was available, we identified Iγ-Cμ and Iα-Cμ CTs, confirmed by sequence analysis (Table 1; data not shown). In normal human circulating B cells, CTs are not detectable in either the naive or memory pool.¹⁵  As 6 of 7 of these MCL samples were peripheral mononuclear cells and 1 was BM derived, these CTs are most likely to be tumor associated. AID expression paralleled CTs in 5 of 5 cases (Figure 2 and Table 1). In normal B cells, CD40L and interleukin 4 (IL-4) induce C_H germ line transcripts.²²  These are also inducible in AID^-/- B cells, but I_H-C_H CTs cannot be generated.^9,10  This indicates that specific CTs identified in MCL represent active CSR events. GC-linked CSR is the normal pathway,^8,9  and this may explain switching in MUT MCL. However, switch events can occur in extrafollicular sites.²³  MCL cells with unmutated V_H genes imply origins from pre-GC B cells and appear to have initiated switch events in a non-GC ectopic environment where somatic mutation is silent. For these cells, the potential exists to respond to T-cell-independent antigens.²⁴ 

When isotype switch to downstream C_H occurs by deletional DNA recombination, Cμ is excised as switch circles.^9,15,22  In these MCL cases, sIgM(D)++ expression is clearly evident and represents the functional allele, with the other allelic 14q32 site fused to the aberrant t(11;14) translocation.^1,2  Fine mapping of both allelic loci by DNA fiber FISH analysis indicates that deletional events in C_H appear to occur infrequently in MCL cells.²⁵  Tumor cells, however, retain the potential to generate switched sIg by recombination on the functional allele. In 1 of 12 cases here, a low number (12%) of tumor cells appears to express sIgA (Table 1), suggesting that in most cases, cell numbers bearing variant sIg are below the detection sensitivity of conventional immunophenotyping in MCL. Identification of mature switch variants by nested RT-PCR, together with specific CTs, indicates that CSR events are occurring, most likely in a tumor subpopulation on the functional allele. This level of switch activity in MCL is not readily identifiable when using generic C_H probes and primary multiplex RT-PCR,⁶  a method that lacks the sensitivity of the tumor-specific CDR3-based primers used here.

Our observations suggest that switch events and AID expression may indeed be uncoupled, at least in some cases, and that AID in MCL is a tumor-related activation phenomenon. This raises the specter of aberrant chromosomal events in MCL.