Histology and Immunohistochemistry
In most cases, low magnification examination of the spleen showed increased white pulp nodules with expansion of the marginal zone (MZ) and concomitant, although variable, neoplastic lymphocytic infiltration of the red pulp (cords and/or sinuses). In a few cases, the red pulp infiltration predominated with minimal tumor load in the white pulp. Finally, in 2 cases, there was complete effacement of both the white and the red pulp by the neoplastic lymphocytic infiltration. In most cases, the neoplastic white pulp nodules showed the “classical” biphasic appearance and were composed of an inner zone of small B lymphocytes surrounded by an outer zone of larger pale cells admixed with a variable amount of B immunoblasts, growing in an MZ pattern with disappearance of the mantle zone. Plasmacytic differentiation was observed in 5 cases.
Neoplastic lymphocytes exhibited a phenotypic profile, which is considered consistent, albeit not specific, with SMZL: CD20+ /CD22+ /CD79a+ /CD5- /CD23− /CD43− /CD10− /CD3− /BCL-6−/BCL-2−/ CCND1 (cyclin D1)− /kappa+ or lambda+. The use of a broad spectrum of monoclonal antibodies against the above-mentioned markers permitted the exclusion of other B-cell lymphoproliferative disorders mimicking SMZL in the spleen, such as B chronic lymphocytic leukemia (CLL), hairy cell leukemia, mantle cell lymphoma, follicular lymphoma, and lymphoplasmacytic lymphoma. All cases tested negative by PCR for either BCL-2/IGH or BCL-1/IGH chimeric genes.
IGHV, IGKV, and IGLV Gene Usage in Individual SMZL Clones
Clonal IGHV-D-J rearrangements were amplified by PCR in 42/43 cases. Four cases had 2 different rearrangements involving a different IGHV gene. The 46 amplified rearrangements used germline IGHV genes of the IGHV1 (7 cases), IGHV3 (17 cases), IGHV4 (19 cases), and IGHV5 subgroup (3 cases). Two out of 44 rearrangements for which complete CDR3 sequence data were available had a stop codon or an out-of-frame rearrangement and, thus, were unproductive; 1/2 unproductive sequences belonged to a case with double rearrangements. Among IGHV3 subgroup genes, no bias was observed in favor of a particular gene. In contrast, IGHV4-34 was detected in 8/19 rearrangements using IGHV4 genes (Table 1). Other frequent genes were IGHV4-61 (4/19 rearrangements), IGHV4-59, and IGHVH4-39 (3/19 rearrangements each).
Information on clonotypic light-chain expression was available in 35/43 cases: 20/43 expressed kappa chain, whereas 15/43 cases expressed lambda chain. Clonal IGKV-J rearrangements were amplified in 17/20 kappa-SMZL cases, 7/15 lambda-SMZL cases (one case with double rearrangements, each with a different IGKV gene; IGKV4-1 [B3], IGKV1-33/1D-33 [O18,O8]), and 6/8 cases for which information on light-chain expression was missing. Nine unproductive IGKV-J sequences were amplified in 1 kappa-SMZL case utilizing the IGKV2-29 (A18) gene with a stop codon instead of cysteine at position 104 in FR3-IMGT), 4/15 lambda cases (one case with a double rearrangement), and 3/7 cases for which information on light-chain expression was missing. The 31 IGKV-J rearrangements analyzed used 14 different germline IGKV genes (Table 2); IGKV4-1 (B3) was the most frequently used IGKV gene (8/31 IGKV sequences), always with a different CDR3 region.
IGKV-KDE rearrangements were amplified in 9/15 cases; assignment of IGKV gene was possible for 6/10 IGKV-KDE rearrangements and revealed usage of the following genes: IGKV1-16 (L1) [2 cases], IGKV2D-28 (A3) [2 cases], IGKV2-30 (A17), IGKV1D-16 (L15) [1 case each]. JKI-KDE rearrangements that would delete the IGKV-J rearrangement (if present) were detected in 6/9 cases analyzed. Taking IGKV-J, IGKV-KDE, and JKI-KDE rearrangements together, 13/15 SMZL cases with clonotypic lambda chain had at least one rearranged IGK locus.
Clonal IGLV-J sequences were amplified by the PCR in 12/15 cases expressing clonotypic lambda chain; one rearrangement was out-of-frame. Nine different germline IGLV genes were recognized in the twelve rearrangements analyzed (24). IGLV1 subgroup genes were used with the highest frequency (5/12 cases) (Table 2).
CDR3 in heavy- and light-chain rearrangements of individual SMZL clones
Analysis of the HCDR3 region was possible in 44/46 rearrangements and revealed that SMZL-HCDR3 composition was comparable to normal PB IgM+ B cells in length (median: 17 aminoacids, range: 8–35), number of N-nucleotides and 5′ and 3′ exonuclease activity at, respectively, the V and J genes. Similar to normal B cells (29), longer HCDR3 regions were observed in SMZL cases with unmutated sequences. The distribution of IGHJ genes was similar to that reported for normal B cells (30): IGHJ4 was the most frequent gene followed by IGHJ6 (23 and 11 sequences, respectively). Most cases used IGHD genes from the IGHD2, IGHD3, and IGHD6 subgroups (6, 19, and 8 sequences, respectively) (31), which are also frequently used by normal B cells (32). Similar to normal peripheral blood B cells (30) as well as tonsillar subepithelial B cells (6), the hydrophilic IGHD gene reading frame (RF) predominated in CDR3 sequences for which IGHD segment assignments could be made. Among unmutated sequences, a more even distribution of the hydrophilic and hydrophobic RFs was observed. No subgroups of cases with homologous HCDR3 sequences were identified.
Complete analysis of the CDR3 region was possible in 28/31 IGK and 12/12 IGL rearrangements. The KCDR3 composition of the analyzed SMZL sequences was comparable to normal PB IgM+ B cells in length (median: 9 aminoacids, range: 7–11), number of N-nucleotides, and 5′ and 3′ exonuclease activity at, respectively, the V and J genes. All possible IGKJ gene segments were recognized. Similar to normal peripheral blood IgM+ B cells (33), IGKJ2 was the most frequent gene (10 cases), followed in order by IGKJ4 (8 cases), IGKJ1 (6 cases), IGKJ3 (3 cases), and IGKJ5 (1 case).
LCDR3 had a median length of 11 aminoacids (range, 9 to 12); N-nucleotide insertion and 5′ and 3′ exonuclease activity at, respectively, the V and J genes, was similar to normal B cells. Similar to the normal repertoire (34–36), the IGLJ2/3 genes were used in most (9/12) IGLV-J rearrangements with complete LCDR3 sequence data.
Somatic Hypermutation Analysis
Twenty-seven IGHV-D-J rearrangements carried mutated IGHV genes (less than 98% homology with the closest germline gene), while the remainder (19/46) had unmutated IGHV genes. IGH-mutated rearrangements most often utilized IGHV3 subgroup genes (14/27 cases). A strong association with IGHV4 subgroup usage was identified in unmutated rearrangements (11/19 cases). Mutations were exclusively single nucleotide substitutions and localized mainly in CDR1, CDR2, and FR3-IMGT (23). Similar to normal peripheral blood B cells (37), follicular lymphoma, and multiple myeloma (38), a higher incidence of mutations in RGYW/WRCY motifs were observed in CDRs rather than in framework regions (FRs)
Among IGHV1 genes, replacement mutations were frequent at IMGT-CDR1 positions 32 (frequency of sequences carrying a replacement mutation at that position: 43%) and 33 (28%) and at IMGT-FR3 positions 87 and 101 (25% each). Nevertheless, mutations at the last 2 positions led to replacement by amino acids of similar charge and size. Among IGHV3 genes, replacement mutations were frequent at IMGT-CDR1 position 32 (70%), IMGT-FR2 position 55 (30%), IMGT-CDR2 positions 56 (30%) and 62 (42%), and at IMGT-FR3 positions 92 and 96 (32% each). Finally, among IGHV4 genes, replacement mutations were frequent at IMGT-FR2 positions 45 (40%) and 48 (30%; all mutations leading to replacement by aminoacids of similar charge and size), IMGT-CDR2 positions 59 (29%; in sharp contrast to IGHV1 and IGHV3 genes with 0% and 10% sequences mutated at that position) and 61 (36%), and IMGT-FR3 positions 78 (29%) and 92 (43%). Compared with IGHV1 and IGHV3 genes, IGHV4 genes had a lower incidence of replacement mutations in IMGT-CDR1. The invariant serine (S)-92 at IMGT-FR3 was mutated in 43/32/0% of IGHV4/3/1 sequences. Frequently mutated serines in IGHV4 genes were also S-59 and S-61 in IMGT-CDR2 (see above). In contrast, other invariant serines among IGHV4 genes were either rarely mutated (for example, S-29, S-31, S-32 in IMGT-CDR1) or never mutated at all (for example, S-16, S-20, S-26 in IMGT-FR1, S-70, S-74, S-79, S-83, S-88, and S-93 in IMGT-FR3). Positions with conserved properties (cysteines at IMGT positions 23 and 104, tryptophane-41, aliphatics at positions 21 and 89, amide-44, proline-46, glycine-47, basic-75, acidic-98 and tyrosine-102 ) as well as tryptophane-52, involved in the VH-VL domain interaction (36), were never found to carry a replacement mutation in the present series. Among IGKV + IGLV genes, only 1 replacement mutation was observed in one of the string of 5 glycines at positions 16, 47, 70, 78, and 84 (39). Mutation analysis after the multinomial distribution model disclosed statistically significant evidence for positive selection by antigen in 5/27 mutated IGHV genes.
In the case of IG light chains, 9/31 IGKV-J and 6/12 IGLV-J rearrangements carried mutated V genes. As in IGHV genes, mutation targeting to the RGYW/WRCY motifs was observed in CDRs rather than FRs. Statistically significant evidence for positive selection by antigen was obtained in 4/9 mutated IGKV and 1/6 mutated IGLV genes.