Immunoglobulin (Ig) Repertoire in Multiple Myeloma: High Frequency of Recurrent Aminoacid Substitutions in the FR2 and CDR2.

Background. Characterization of the heavy chain (IgH) repertoire and assessment of rate and nature of somatic mutations (SM) provided important clues to understand the pathogenesis of CLL and other lymphoid tumors. The IgH repertoire of MM has been studied less extensively. This aspect has been analyzed in a large population of Italian MM patients. The results have been compared to published repertoires from normal subjects (Brezinschek et al J Immunol 1995) and to our bank of more than 200 CLL.

Methods. MM-specific Ig heavy chain rearrangements were sequenced as described elsewhere (Ladetto et al BBMT 2000) starting from 220 patients. The analysis was successful in 162 with a close-to-expected success rate. IgH sequences were analyzed using the Basic local alignment search toll (BLAST), (http://www.ncbi.nlm.nih.gov/igblast) to identify V, D and J regions and to establish the rate of somatic mutations and their silent to replacing (S/R) ratio. Finally the aminoacid sequence derived from the IgH rearrangement was analyzed to identify recurrent aminoacid substitutions. The clinical features of our patients are: age median 55 years (34–73), male sex 53%, Stage III MM 61%, IgG MM 53%, IgA MM 24%, light chain MM 17%.

Results. Most VH families were not over- or under-represented compared to normal controls. The only family strongly over-represented in MM compared to both CLL and normal repertoires was the VH 3–33 (22% of patients) (p<0.0001). In addition, some families were under-represented (VH 3–49,VH 4–59, VH4–31) compared to normal subjects (p<0.05). Three D families were under-representred (DH 2–2, DH 5–5 DH 6–13) and two over-represented (DH 4–17, DH 5–24) (p<0.05). JH usage was not skewed in MM. The median rate of VH SM in MM was 5.3% (0.5%–18.8%). When the position and type of aminoacid replacements were evaluated a striking number of recurrent substitutions were noticed. In three families (VH 3–21, 3–23 and 3–33) recurrent mutations occurring in more than 40% of cases were noticed. In family 3–21, 6 of 10 patients (60%) had an identical triple aminoacid substitution in CDR2 at position 5 (S to D), 6 (S to G) and 8 (Y to N). In the VH 3–23 a S to D substitution at position 5 and a S to N substitution at position 8 of the CDR2 occurred in 11 (44%) and 10 (40%) of 25 patients, without apparent association or mutual exclusion. Finally, in the over-represented VH 3–33 family an A to S mutation involving the FR2 (position 14) occurred in 25 of 36 patients (69.4%) and a W to S mutation in the CDR2 at position 3 occurred in 24 patients (66.7%). The two mutations co-existed in all but three cases. These mutations were much less common in VH-mutated sequences of CLL (median rate: 20% range 9–36%) and normal repertoires (median 0%, range 0–28%) despite a comparable overall rate of SM. These differences reached statistical significance for all but one mutation. Moreover the recurrent triple and double mutation pattern of VH3–21 and VH 3–33 were observed only occasionally in non-MM populations.

Conclusions. This extensive analysis on Ig repertoire in MM suggest that malignant plasmacells derive from lymphocytes that underwent specific pathways of antigen selection which might be relevant for MM pathogenesis. Additional studies are required to address epidemiological aspects (VH3–33 over-representation was not seen in previous smaller studies from other countries) and to identify potential responsible antigens.