Matutes, Estella

Abstract We describe the features of 24 cases of atypical CD5+ small B-cell leukaemia unclassifiable by current methods. The male/female ratio was 3 (18M/6F), with a median age at diagnosis of 58 years (range 39–84 years). All cases were CD5 positive, but with an immunophenotype and morphology atypical for CLL. All cases were negative for t(11;14) and other B cell malignancies were excluded. FISH analysis was carried out using probes for IGH break apart, t(14;19) involving BCL3, trisomy 12, 13q14, 11q22–23 (ATM) and 17p13 (p53 locus). IgVH somatic mutations were analysed using direct sequencing (2% cut off). FISH using IgH break apart probe showed 8 (32%) cases with a split at 14q32. These 8 cases were further investigated using a FISH probe for t(14;19). Four cases (16%) exhibited t(14;19) ie. showed the IgH/BCL3 rearrangement in >70% of cells. Further characterisation of the 24 cases, at additional loci showed that 4 (16%) patients had 17p deletion, 3 (12%) 11q deletion, 3 (12%) 13q deletion, and 14 cases (56%) trisomy 12. The 8 cases showing a split IgH signal did not show deletions of 17p, 11q, or 13q but trisomy 12 was present in 7/8 (88%) patients. Sixteen out of 24 (67%) cases had unmutated IgVH genes with VH3 and VH4 families being used in 91% (22/24). Interestingly, unlike in chronic lymphocytic leukaemia (CLL) and mantle cell lymphoma (MCL), the VH1 family was not used in this group of patients. All 4 cases with t(14;19) showed unmutated IgVH genes. Of the sixteen patients without involvement of 14q32, only 2 patients have died with a median follow up of 5.5 years. Of the 4 cases with the t(14;19) (IgH/BCL3 rearrangement), 3 required treatment within 2.5 years of diagnosis and two out of four patients died 5 years after diagnosis, following Richter’s transformation suggesting that this rearrangement is a poor prognostic factor and defines a group with a distinct clinical outcome. Our findings show a high frequency of 14q abnormalities in atypical CD5+ small B-cell leukaemia. BCL3 rearrangement is seen in a significant subset of these cases. The BCL3 gene encodes a nuclear protein that belongs to the IkB family of inhibitors of NFkB. It is involved in the regulation of a variety of transcription factors and it is able to stimulate the expression of cyclin D1. The dysregulation of BCL3 via its juxtaposition to the regulatory element of the IgH locus in our cases suggests a pathogenic role for BCL3 in these B cell disorders and may define a group of patients with a more aggressive clinical course. Further, we have examined these cases for signatures, defined in myeloma, characteristic of NFkB deregulation. We suggest that in cases of CD5 positive B-cell disorders with an immunophenotype and morphology atypical for CLL, and negative for t(11;14), rearrangements involving the IgH locus are frequent and have allowed us to identify cases with poor prognosis and with a pathogenesis that involves deregulation of NFkB.

Abstract Abstract 2638 We recently demonstrated that over 30% of cases with splenic marginal-zone lymphoma (SMZL) express distinctive immunoglobulin (IG) receptors that utilize a single polymorphic variant of the IGHV1-2 gene (IGHV1-2*04) and also exhibit restricted antigen-binding site motifs and precise targeting of somatic hypermutation (SHM). On these grounds, we proposed the existence of molecular subtypes of SMZL defined by immunogenetic analysis of the IG receptors with implications for selection by specific (super) antigenic element(s) in the development of at least a major subset of SMZL. In order to gain insight as to whether antigen involvement is relevant only prior to the malignant transformation or if it continues to affect the SMZL clone itself leading to intraclonal diversification (ID) through ongoing SHM, we conducted a large-scale subcloning study of rearranged IG heavy variable genes, in a total of 471 subcloned sequences from 22 SMZL cases. The analysis was intentionally biased towards cases expressing IGHV1-2*04 receptors that exhibit a series of distinctive immunogenetic features, including biased usage of the IGHD3-3 and IGHD3-10 genes, unusually long heavy complementarity-determining region 3 (VH CDR3) and minimally/borderline mutated status (identity to the germline in the range of 97–99.6%). Hence, the study group included 16 IGHV1-2*04 cases and 6 cases utilizing other IGHV genes. PCR reactions were run using the high-fidelity Accuprime Pfx polymerase and a median of 21 (9–46) colonies/case were analyzed. All “non-ubiquitous” sequence changes from the germline were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence from the same sample; (ii) confirmed mutation (CM) - a mutation observed in more than one but in less than all subcloned sequences from the same sample. Overall, 15/22 cases (68%) carried intraclonally diversified IGHV-D-J genes with CMs amongst subclones, of which 12 utilized the IGHV1-2*04 gene whereas the remaining 3 utilized other IGHV genes. The high frequency of ID within the IGHV1-2*04 group, ranging from limited to (often) pronounced, is noteworthy in view of the generally low level of SHM among IGHV1-2*04 receptors. Detailed analysis of the distribution and molecular features of CMs revealed: (i) restricted ID patterns, in the sense of identical mutations in certain VH positions among subclones of different cases; (ii) “hotspots” of ID, i.e. particular codons exhibiting intense ongoing mutational activity (especially notable in this respect was codon 39 in VH FR2); (iii) a predominance of conservative amino acid changes, characterized by somatically introduced amino acid belonging to the same biochemical category as the mutating amino acid; and (iv) limited diversification within VH CDR3. Additionally, in 7/12 IGHV1-2*04 cases with CMs, identified ID patterns delineate distinct “clusters” of subcloned sequences with unique as well as shared mutations and closely similar if not identical VH CDR3s (including identical VH CDR3 length), pointing to “branching” of the malignant clone into distinct subclones, perhaps able to evolve along related yet distinct pathways. In conclusion, our study indicates that the SHM mechanism may continuously operate in certain subsets of SMZL, especially those expressing IGHV1-2*04 receptors. Although the precise timing of interactions with antigen(s) and their functional implications for SMZL evolution will likely remain difficult to define accurately, the results reported here suggest a role for persistent antigenic stimulation, at least for certain immunogenetically defined subsets of SMZL. Disclosures: Mollejo: Red Temática de Investigación Cooperativa en Cancer (RETICC): Research Funding; Asociación Española contra el Cancer: Research Funding.

Abstract Abstract 634 We systematically explored the immunoglobulin (IG) gene repertoire in 337 cases with splenic marginal-zone lymphoma (SMZL), by far the largest series yet. To resolve classification uncertainties, we included in the analysis only cases with a diagnosis of SMZL based on spleen histopathological findings or cases fulfilling the 2008 SBLG criteria (Matutes et al. Leukemia 2008). We here report that the IG heavy variable (IGHV) gene repertoire in SMZL is remarkably biased, with only three genes accounting for 45.8% of cases (IGHV1-2, 24.9%; IGHV4-34, 12.8%; IGHV3-23: 8.1%, respectively), significantly extending previous similar observations. Particularly for the IGHV1-2 gene, strong biases became evident at the level of utilization of different alleles, since 79/86 rearrangements (92%) utilized allele *04 vs. only 7/86 rearrangements (8%) that utilized allele *02. This is noteworthy, taking into consideration that these two alleles differ in a single nucleotide, leading to a single amino acid change in framework region (FR)-3. The repertoire biases became more pronounced when the analysis was focused on 171 rearrangements from 163 cases classified as SMZL based on splenic histopathology, according to the 2008 WHO criteria. Within this subgroup, 56/171 cases (32.7%) utilized IGHV1-2*04. Noticeably, only 1/17 cases with a diagnosis of splenic diffuse red pulp lymphoma utilized IGHV1-2*04 (p<0.02 for comparison to SMZL). The IGHV1-2*04 rearrangements carried significantly longer heavy complementarity-determining region-3 (VH CDR3) than all other cases (median, 22 vs. 17 amino acids, respectively; p<0.001). In addition, 52/79 IGHV1-2*04 cases (65.8%) employed one of the IGHD3-3, IGHD3-9 or IGHD3-10 genes. In 28/32 IGHV1-2*04/IGHD3 rearrangements, the IGHD gene was utilized in the same reading frame, leading to VH CDR3s with common “IGHD-derived” amino acid (AA) motifs. Using bioinformatics tools previously applied to CLL, biased associations of IGHV, IGHD and IGHJ genes with stereotyped VH CDR3s were identified in 25/345 sequences (7.2%). Noticeably, only 10/28 IGHV1-2*04/IGHD3-3 rearrangements with “IGHD-derived” VH CDR3 amino acid motifs could be assigned to “stereotyped” clusters. Despite exhibiting restricted usage of the IGHV1-2*04 and IGHD3-3 genes leading to great overall VH domain similarity, the remaining cases did not fulfill the established criteria for VH CDR3 “stereotypy”, as defined in other lymphoid malignancies, namely CLL. Based on somatic hypermutation (SHM) analysis, the sequences were divided into three groups: (i) truly unmutated (100% germline identity, GI): 46/345 sequences (13.3%); (ii) minimally/borderline mutated (97-99.9% GI): 130/345 sequences (37.7%); and (iii) significantly mutated (<97% identity): 169/345 sequences (49%). At the individual gene level, the distribution of rearrangements of IGHV genes according to SHM status varied significantly. In particular, 56/79 IGHV1-2*04 rearrangements (71%) were predominantly “borderline mutated”, whereas the majority (>67%) of rearrangements utilizing the IGHV3-23, IGHV3-30 and IGHV3-7 genes were “significantly mutated”; finally, IGHV4-34 gene rearrangements were evenly distributed to the three mutational subgroups. Shared (“stereotyped”) AA changes were identified for IGHV1-2*04 rearrangements, with certain FR2 and FR3 codons emerging as “hotspots” for recurrent, conservative AA changes. In conclusion, we demonstrate that more than 30% of cases with a histopathological diagnosis of SMZL on the spleen express IGHV1-2*04 receptors with unusually long VH CDR3s, biased usage of the IGHD3-3 gene, leading to shared “IGHD-derived” VH CDR3 motifs, and very precise molecular features of SHM. The biased expression of a distinctive germline-encoded VH specificity might be considered as evidence for heavy chain dominance in the clonogenic IG receptors in SMZL. These findings allude to selection by specific (super)antigenic element(s) in the pathogenesis of at least a major subset of SMZL. In addition, they raise the intriguing possibility that certain subtypes of SMZL could derive from progenitor cell populations adapted to particular antigenic challenges through cellular selection of VH domain specificities. Disclosures: No relevant conflicts of interest to declare.

Abstract We performed a meta-analysis of 3 genome-wide association studies to identify additional common variants influencing chronic lymphocytic leukemia (CLL) risk. The discovery phase was composed of genome-wide association study data from 1121 cases and 3745 controls. Replication analysis was performed in 861 cases and 2033 controls. We identified a novel CLL risk locus at 6p21.33 (rs210142; intronic to the BAK1 gene, BCL2 antagonist killer 1; P = 9.47 × 10−16). A strong relationship between risk genotype and reduced BAK1 expression was shown in lymphoblastoid cell lines. This finding provides additional support for polygenic inheritance to CLL and provides further insight into the biologic basis of disease development.