Clonal Selection in the Ontogeny and Evolution of Splenic Marginal Zone Lymphoma Confirming the Existence of Distinct Molecular Subtypes

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1556-1556
Author(s):  
Vasilis Bikos ◽  
Evangelia Stalika ◽  
Maria Karypidou ◽  
Panagiotis Baliakas ◽  
Zadie Davis ◽  
...  
Keyword(s):  
Amino Acid ◽  
Light Chain ◽  
Marginal Zone ◽  
Molecular Subtypes ◽  
Conflicts Of Interest ◽  
Clonal Selection ◽  
Marginal Zone Lymphoma ◽  
Biological Research ◽  
Splenic Marginal Zone Lymphoma ◽  
Light Chain Gene

Abstract Abstract 1556 Splenic marginal-zone lymphoma (SMZL) exhibits biased immunoglobulin (IG) heavy and light chain gene repertoires, alluding to selection by antigens and/or superantigens in lymphomagenesis. We recently reported that immunogenetic analysis of the IG receptors enables the identification of molecular subtypes of SMZL, since, remarkably, greater than 30% of cases can be assigned to a single subgroup defined by usage of the *04 polymorphic variant of the IGHV1–2 gene. The IGHV1–2*04 receptors carry long and often positively charged heavy complementarity-determining region 3 with restricted motifs and show biased associations with certain light chain genes (IGKV3–20, IGKV1–8, IGLV2–14). Furthermore, they exhibit low-level, yet non-randomly targeted somatic hypermutation (SHM), likely reflecting a distinctive process of immune interaction with antigen(s). Preliminary work from our group indicated a continued influence of antigen on SMZL clones expressing IGHV1–2*04 receptors reflected in intraclonal diversification (ID) of rearranged IG heavy variable genes. Here we significantly extend our ID studies and explore whether IGHV1–2*04 SMZL are distinctive also in terms of ID compared to SMZL utilizing other IGHV genes. To this end, we performed a comprehensive subcloning analysis of IGHV-IGHD-IGHJ gene rearrangements in a total of 728 subcloned sequences from 39 SMZL cases, including 20 cases expressing IGHV1–2*04 receptors with a median identity to the germline (GI) of 98.7% and 19 cases utilizing other IGHV genes, namely IGHV1–18 (n=3), IGHV3–23 (n=6), IGHV3–30 (n=30), IGHV4–34 (n=3), IGHV4-4 (n=1) and IGHV5–51 (n=3), with a median GI of 97.9%. 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. In order to compare mutation counts between different rearrangements, mutations were normalized to both the nucleotide length and the number of subcloned sequences for each rearrangement. ID was identified in 17/20 (85%) IGHV1–2*04 rearrangements, with confirmed nucleotide substitutions ranging from 1 to 21 per case for a total of 77 unique substitutions. Of these, 51 resulted in the replacement of the germline-encoded amino acid (Replacement mutations, R), whereas the remaining 26 were silent (S). Certain codons (e.g: VH CDR1–36, VH FR2–39, VH FR3–87) were highly targeted for novel changes leading to ID. Interestingly, identical confirmed mutations were shared by subclones of different rearrangements confirming a remarkable restriction in the ongoing SHM profiles in that subclones shared identical confirmed mutations. In fact, 12 amino acid replacements were confirmed in the subclones of at least two rearrangements and were characterized as ”recurrent”. In contrast to IGHV1–2*04 rearrangements, only 8/19 (42%) rearrangements utilizing other IGHV genes showed imprints of ID (p=0.005). Moreover, the ID profiles of non-IGHV1–2*4 cases were different, as evidenced by both the lower numbers of confirmed mutations and the lack of shared mutations (per group of cases utilizing the same IGHV gene). In conclusion, continuous stimulation by antigen seems to be relevant not only for SMZL development but also for selection leading to clonal evolution, at least for a substantial proportion of cases. The extensive and distinctive ID observed in IGHV1–2*04 cases compared to cases utilizing other IGHV genes further supports the hypothesis that the former might constitute a distinct subgroup with important implications for both SMZL sub-classification and future clinical and biological research. Disclosures: No relevant conflicts of interest to declare.

Absence of Mutations of the Histone Methyltransferase Gene EZH2 In Splenic B-Cell Marginal Zone Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5086-5086
Author(s):  
Luz Martínez-Avilés ◽  
Marta Salido ◽  
Beatriz Bellosillo ◽  
Vera Adema ◽  
Ana Ferrer ◽  
...  
Keyword(s):  
B Cell ◽  
Marginal Zone ◽  
Mutational Analysis ◽  
Conflicts Of Interest ◽  
Direct Sequencing ◽  
Normal Karyotype ◽  
Marginal Zone Lymphoma ◽  
Low Grade ◽  
Splenic Marginal Zone Lymphoma ◽  
7Q Deletion

Abstract Abstract 5086 Background Splenic marginal zone lymphoma (SMZL) is a rare low-grade B-cell lymphoproliferative disorder with characteristic clinical, cytological, histological and immunophenotypical features. The most common cytogenetic abnormality, present in 30–40% of the patients is the 7q deletion, that extends from 7q21 to 7q36. This aberration may represent a primary pathogenic event in SMZL. Recently, mutations in the EZH2 gene, located at 7q36.1, have been described in different hematological malignancies including B-cell lymphomas. However, the role of the EZH2 gene in SMZL has to be elucidated. Aim To determine the prevalence of EZH2 mutations in a cohort of SMZL patients. Patients and Methods Twenty-nine patients with SMZL were screened for mutations in the EZH2 gene. From the whole cohort, 11 patients presented 7q deletion (three of them as a single anomaly), 11 had a normal karyotype and 7 had other cytogenetic aberrations. The mutational analysis of the EZH2 gene was performed by direct sequencing using primers covering the whole exome of the gene. DNA was extracted from CD19 isolated B-cells from peripheral blood or from total lymphocytes if the percentage of pathologic B-cell was higher than 50%. Results From the whole cohort of 29 SMZL patients, no pathogenic mutations (frameshift or nonsense mutations) were detected in the EZH2 gene in any of the patients analyzed. Five patients harboured the missense mutation D185H in exon 6, that has been previously described as a single nucleotide polymorphism (SNP). Conclusions In conclusion, the EZH2 gene is not mutated in our series of SMZL patients suggesting that this gene is not involved in the pathogeny of this entity. Acknowledgments: Fellowship FI2008 (AGAUR) to LMA, This work was supported (in part) by grants from Instituto de Salud Carlos III FEDER; Red Temática de Investigación Cooperativa en Cáncer (RTICC, FEDER): RD06/0020/0031 and RD07/0020/2004; Ministerio de Sanidad y Consumo (Spain): PI07/0586. Disclosures: No relevant conflicts of interest to declare.

Over 30% of Patients with Splenic Marginal Zone Lymphoma Express Distinctive Antigen Receptors Utilizing a Single Immunoglogulin Variable Gene: Implications for the Origin and Selection of the Neoplastic Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 634-634 ◽  
Author(s):  
Vasilis Bikos ◽  
Nikos Darzentas ◽  
Anastasia Hadzidimitriou ◽  
Zadie Davis ◽  
Sarah Hockley ◽  
...  
Keyword(s):  
Amino Acid ◽  
Marginal Zone ◽  
Marginal Zone Lymphoma ◽  
Single Amino Acid ◽  
Histopathological Diagnosis ◽  
Splenic Marginal Zone Lymphoma ◽  
Gene Repertoire ◽  
Single Amino Acid Change ◽  
Vh Domain ◽  
Selection Of

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.

Splenectomy Is Still a Valid Option For Splenic Marginal Zone Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5129-5129
Author(s):  
Alessandro Pulsoni ◽  
Pasqualina D'Urso ◽  
Gianna Maria D'Elia ◽  
Giorgia Annechini ◽  
Caterina Stefanizzi ◽  
...  
Keyword(s):  
Marginal Zone ◽  
Conflicts Of Interest ◽  
B Cell Lymphoma ◽  
Marginal Zone Lymphoma ◽  
Splenic Marginal Zone Lymphoma ◽  
Spleen Size ◽  
Second Line ◽  
Second Line Therapy ◽  
Line Therapy

Abstract Introduction Splenic marginal zone lymphoma (SMZL) is an indolent B-cell lymphoma characterized by splenomegaly, frequent moderate lymphocytosis with or without villous morphology and possible involvement of various organs, especially the bone marrow (BM). Diagnosis is classically based on the spleen histology, but it can be made on the BM biopsy, based on the typical intrasinusoidal cell infiltration pattern and immunohistochemistry. Different therapeutic options are available, but to date there are no conclusive comparative data. Patients and methods We retrospectively analyzed 83 consecutive patients with a diagnosis of SMZL observed at our Institution between 1999 and 2013. The diagnosis was based on the BM biopsy in 79 patients; the BM was negative in 4 patients. Diagnosis was histologically confirmed on the spleen in 27 patients who underwent splenectomy. Patients presented a median age of 72.5 years (range 38-84); 43 were males. The median spleen size at diagnosis was 145 mm, ranging from 100 to 300 mm. The majority of patients were stage IV at diagnosis for BM infiltration (95%); B symptoms were present in 4 of them (4.8%). Forty-two patients (50.6%) had a lymphocytosis at diagnosis and 13 (15.6%) presented an IPI score higher than 3. Thirty-five of them (42%) had a MZL BM infiltration superior to 30% of the total bone cellularity. Forty-two patients (50.6%) underwent a watch and wait policy (WW), while 41 (49.4%) were treated within 6 months from diagnosis, mainly because of symptomatic splenomegaly; in these patients, treatment consisted of splenectomy, chemotherapy or chemotherapy plus immunotherapy with Rituximab. The features of patients submitted to WW with respect to patients treated at diagnosis were comparable for the various parameters mentioned above, except for spleen size (higher in patients treated at presentation) and lymphocyte count (higher in patients who were observed). After a median follow-up of 64 months, the overall median survival was 96%. Among the 42 WW patients, 18 (42.8%) are still untreated after a median follow-up of 57.5 months, while 24 (57.2%) have required therapy; the median treatment-free interval in these patients was 25.5 months. Concerning the 41 patients who underwent treatment at diagnosis, after a median follow-up of 50 months, 13 (31.7%) have required a subsequent second-line treatment. The interval between first-line approach and re-treatment in patients treated at diagnosis was 30 months. Overall, 27 patients were treated with splenectomy only (either at diagnosis or after a WW period): only 6 of them (22%) had a subsequent progression after a median latency of 42 months; 26 were treated with chemotherapy alone (alkylating agents in the majority of them, combination therapy in a minority): 15 of them (60%) had a subsequent relapse or progression after a median of 9 months; 12 patients received a Rituximab-containing regimen: of these, only 2 (16%) have so far required a second-line therapy after 10 and 26 months respectively. Conclusions The WW policy is a valid option for asymptomatic patients: in these patients, after 4.5 years from diagnosis more than 40% is still untreated. In patients requiring treatment, splenectomy alone is followed in the majority of patients by a long period of good disease control: only 22% required a second-line therapy after 3.5 years. The addiction of Rituximab to chemotherapy seems to reduce the probability of relapse and to prolong the response duration. However, these preliminary data need to be confirmed by larger studies. Disclosures: No relevant conflicts of interest to declare.

Monoclonal B-Cell Lymphocytosis Exhibiting Immunophenotypic Features Consistent with Marginal Zone Origin: What Is This Entity?

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1587-1587
Author(s):  
Aliki Xochelli ◽  
Panagiotis Baliakas ◽  
Anne Gardiner ◽  
Sarah Mould ◽  
Zadie Davis ◽  
...  
Keyword(s):  
B Cell ◽  
Marginal Zone ◽  
Conflicts Of Interest ◽  
Single Case ◽  
B Cell Lymphoma ◽  
Marginal Zone Lymphoma ◽  
Gastric Malt Lymphoma ◽  
Splenic Marginal Zone Lymphoma ◽  
Group A ◽  
Immunogenetic Analysis

Abstract Abstract 1587 Monoclonal B-cell lymphocytosis (MBL) with an immunophenotype consistent with marginal zone origin (MBL-MZ), that can be either CD5− or CD5+ but atypical for CLL, and also lacking an IGH/CCND1 translocation, is an increasingly recognised entity with poorly understood biological background and clinical significance. In particular, it is not yet clarified whether it represents a precursor state to one of the distinct lymphoma entities recognized by WHO as deriving from MZ cells or whether it constitutes a novel entity, likely with similar ontogeny. To obtain insight into this issue we retrospectively evaluated a series of 71 patients (male/female: 35/36, median age: 73.3 years) with lymphocytosis (median lymphocyte count: 5.77 × 109/l) detected incidentally on a routine blood test. No case had lymphadenopathy, organomegaly or any clinical features to suggest a concurrent marginal zone lymphoma. Hemoglobin and platelet counts were normal in all cases; 15/57 (26%) cases had paraproteinemia. Peripheral blood immunophenotyping revealed the presence of a clonal B-cell population with Matutes score <2 in all cases. Individual markers were expressed as follows: CD5: 15/71, CD23: 7/70, CD79β: 60/64, FMC7: 50/67, CD49d: 35/35, CD38: 6/53. In 8/45 cases assessed with CT-scan and/or ultrasound, borderline splenomegaly was observed. Histopathological examination of the bone marrow biopsy (BMB) was available in 11 cases and demonstrated mostly mixed patterns of neoplastic lymphocytic inflitration from small B cells. Karyotype data were available in 66 cases; 48/66 (72.7%) had abnormal karyotype. Main cytogenetic findings are as follows: (1) translocations: n=16 cases of which 3 carried t(2;7)(p11;q21/22); (2) isochromosome 17q: n=8; (3) trisomy 12: n=8; (4) del(7q): n=7; (5) trisomy 3: n=4. Immunogenetic analysis revealed overusage of the IGHV4–34 gene (15/63, 23.8%). Notably, the IGHV1–2 gene was utilized by a single case, thus sharply contrasting (p<0.0001) splenic marginal-zone lymphoma (SMZL) with a reported frequency of IGHV1–2 in excess of 30%. Seven of 63 rearrangements (11.1%) carried IGHV genes with no somatic mutations, whereas the remainder (56/63, 88.9%) exhibited some impact of somatic hypermutation (SHM), ranging from minimal to (mostly) pronounced. Overall, cases of the present study exhibited a significantly (p<0.005) higher SHM load compared to SMZL. With a median follow-up of 4.9 years (0.8–20), 54 cases (group A) remain stable with no signs of progression. The remaining 17 cases (group B) have MBL along with clinical or histopathologic evidence of lymphoma. In particular: (i) a female with MBL as an incidental finding, also carrying t(2;7), was eventually diagnosed with gastric MALT lymphoma at +11 months from presentation; immunogenetic analysis confirmed clonal identity between the MBL and the lymphoma; (ii) 2 cases developed lymphadenopathy; (iii) a single case developed diffuse large B cell lymphoma of the skin; and, (iv) 13 cases developed splenomegaly and, thus, can be considered as either SMZL or splenic lymphoma/leukemia unclassifiable (SLLU). Groups A and B did not differ in terms of demographics, diagnostic blood counts (including clonal MBL count) and SHM status; the only difference concerned cytogenetic profiles, with i(17)(q10) and del(7q) being almost exclusive to group A. In conclusion, we demonstrate that MBL-MZ can be the presenting feature of occult MZ lymphoma, most frequently SMZL/SLLU. However, in a sizeable proportion of cases, MBL-MZ remains stable over time with no evidence of organ involvement and distinct immunogenetic features from SMZL, thereby raising the possibility that it might represent a newcomer to the spectrum of B-cell lymphoproliferations of MZ origin. Disclosures: No relevant conflicts of interest to declare.

Liver Infiltration In Splenic Marginal Zone Lymphoma: Prevalence and Prognostic Implication

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4137-4137 ◽  
Author(s):  
Eva Domingo-Domenech ◽  
Vicente Romagosa ◽  
Eva González-Barca ◽  
Marta Rodriguez-Aliberas ◽  
Ana Oliveira ◽  
...  
Keyword(s):  
Overall Survival ◽  
Liver Biopsy ◽  
Marginal Zone ◽  
Conflicts Of Interest ◽  
Marginal Zone Lymphoma ◽  
Splenic Marginal Zone Lymphoma ◽  
Prognostic Implication ◽  
Number Of Patients ◽  
Almost All ◽  
Baseline Demographic

Abstract Abstract 4137 Introduction: There are no studies in the literature about the frequency of liver infiltration by splenic marginal zone lymphoma (SMZL). In the last review published by the International Group of SMZL (Matutes E et al, Leukemia 2008;22:487) clinical liver infiltration seems to be infrequent and it is considered, as other organ involvement, to appear during the course of the disease and confers a bad prognosis. Objective: To investigate in a selected sample of patients with SMZL the prevalence of liver infiltration, as well as its possible prognostic implication in survival or transformation. Material and methods: In the majority of our SMZL patients who underwent a splenectomy between 1995 and 2001, a liver biopsy was realised during the surgical procedure in order to investigate hepatic infiltration by lymphoma. Results: Forty-eight patients with SMZL were diagnosed in our center during this period; of them, 19 were splenectomized being liver biopsy performed in 16 (only in those cases that gave informed consent). Splenectomy was performed at diagnosis in 11 patients (time from diagnosis to splenectomy < 3 months). Liver biopsy was positive for SMZL infiltration in 81.3% (13/16) of the cases. Baseline demographic and clinical characteristics of both groups of patients are shown in the following table; there were no significant differences between the groups. We neither find statistical significant differences in overall survival between both groups: mean overall survival for positive and negative liver biopsy patients was 116 ± SD 13 months and 74 ± 29 months, respectively (p=0.69). Conclusion: Histological hepatic infiltration is present in almost all SMZL patients when splenectomized. In our experience, this finding has no prognostic implication. Due to the small number of patients of this study, its significance is limited but to the best of our knowledge, it is the first report that investigates the prevalence of liver infiltration in SMZL and its impact in survival. Larger series are necessary to confirm our findings. Disclosures: No relevant conflicts of interest to declare.

Hierarchical Clustering of B-Cell Receptor Structures in Splenic Marginal Zone Lymphoma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1585-1585
Author(s):  
Paolo Marcatili ◽  
Silvia Zibellini ◽  
Sara Rattotti ◽  
Anna Chailyan ◽  
Marzia Varettoni ◽  
...  
Keyword(s):  
B Cell ◽  
Light Chain ◽  
Binding Sites ◽  
Marginal Zone ◽  
Marginal Zone Lymphoma ◽  
Splenic Marginal Zone Lymphoma ◽  
Sequence Identity ◽  
Role Of Light ◽  
Variable Genes

Abstract Abstract 1585 Splenic marginal zone lymphoma (SMZL) is frequently associated with HCV infection and autoimmune disorders. Previous studies demonstrated a biased usage of immunoglobulin heavy variable genes (IGHV) and, in some cases, stereotyped B-cell receptors (BCRs). This characterization, however, is mainly based on the heavy chain alone, even if strong evidences are emerging on the role of light chain (Bikos et al. Leukemia 2012). The aim of this study was to analyze IG light variable genes (IGLV) of SMZL BCRs, VL-VH pairing and structural information and to investigate the sequence-structure-antigen (AG) relationship. To this end, we analyzed the VL-VH paired sequences of BCR from 52 SMZL pts (38 BM and 14 PB) diagnosed according to Matutes criteria (Leukemia, 2008). Sequences were analyzed using the IMGT/DBs and the IMGT/V-QUEST tool. The PIGS web server was used to build 3-D models of all antibodies (Abs). The Ab structures were compared using LGA and clustered together according to a score accounting for structure and sequence similarity. Using the DIGIT DB and tools, all the clusters were analyzed and compared to other IGs. Based on the IGHV nucleotide sequence identity to the germline, 7 sequences (13%) were considered ‘truly unmutated’ (100% sequence identity), 20 (39%) were ‘minimally or borderline mutated’ (97–99.9%) whereas 25 (48%) were ‘significantly mutated’ (<97%). IGHV families were used as follows: IGHV3 (58%), IGHV1 (27%) and IGHV4 (15%). The majority of pts carried kappa light chain (69%). The most frequently used IGKV families were IGKV3 (58%) and IGKV1 (28%), the most frequent IGLV family was IGLV1 (56%). Considering the VL-VH paired sequences, the two pairings IGHV3-23/IGKV3–20 (n=6) and IGHV1-02/IGLV1-47 (n=3) were significantly over-represented when compared to CLL and DIGIT DB sequences, indicating that the pairing between VL-VH chains was non-random. The IGHV1-02/IGLV1-47 paired sequences showed a high number of somatic mutations (>3%), whereas samples using the IGHV1-02 gene (n=10) but a VL gene other than from IGLV1-47 displayed a low number of mutations, suggesting a significant role for the light chain. In order to analyze the possible functional role of light chain, we analyzed the structural similarity of AG binding sites (ABSs), performing hierarchical clustering on the similarity obtained by an all-against-all structural superposition of each ABS. Twenty structural clusters were identified (8 with ≥3 samples) (Fig. 1). Considering IGs in the same major groups, they showed a similar mutation rate, pointing out a likely common AG selection at least in a fraction of pts (Fig. 1). In most cases, IGs in the same clusters display ABSs with similar physicochemical characteristics: positively charged binding sites (2 clusters), hydrophobic patches (3 clusters) or small pockets in the middle of the ABS (3 clusters) might be clue for different AGs specific for each cluster. HCV infection was found in 1 major and 2 minor clusters (Fig. 1), mainly associated with unmutated clones, indicating a likely common antigenic stimulation. In the other major clusters, the role for an AG-driven selection different from HCV in SMZL lymphomagenesis can be postulated. In particular, 3 clusters, containing both mutated and unmutated samples, displayed a statistically significant similarity to CLL clones (p<0.05), and 1 cluster was structurally similar to autoimmune clones (Kawasaki disease) (p=0.05). Of note, other clusters showed a degree of similarity with samples connected to diseases that involve an AG independent or superantigenic stimulation (EBV, Rabies virus, Rotavirus). In conclusion, the multi-layered characterization of the sequence and structure properties of paired VL-VH in SMZL identified a non-random pairing between heavy and light chains. Structural cluster analysis identified Abs with similar physicochemical properties, similar mutation rate and similar HCV status in a fraction of our dataset. Comparing Abs of our cases to a large dataset of human annotated Abs derived from the DIGIT DB, a subset resulted similar to CLL or autoimmune clones, whereas other Abs appeared more similar to polyreactive Abs and to Abs possibly targeted by superantigens. These findings could explain the large diversity observed in the IGs expressed in SMZL and provide new insights in SMZL pathogenesis. *The first two authors equally contributed to this paper Disclosures: No relevant conflicts of interest to declare.

Ongoing Antigen Interactions In Splenic Marginal Zone Lymphoma: Revelations From The Analysis Of Intraclonal Diversification In Immunoglobulin Light Chain Genes

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2999-2999
Author(s):  
Maria Karypidou ◽  
Evangelia Stalika ◽  
Panagiotis Baliakas ◽  
Vasilis Bikos ◽  
Zadie Davis ◽  
...  
Keyword(s):  
Marginal Zone ◽  
Conflicts Of Interest ◽  
Marginal Zone Lymphoma ◽  
Molecular Evidence ◽  
Splenic Marginal Zone Lymphoma ◽  
Gene Repertoire ◽  
Nucleotide Substitutions ◽  
Time Points ◽  
Molecular Features ◽  
Mutational Status

Abstract Immunogenetic studies have made seminal contributions towards understanding the pathogenesis of splenic marginal zone lymphoma (SMZL) by documenting a highly skewed immunoglobulin (IG) gene repertoire with molecular features strongly implicating selection by antigen(s) in disease ontogeny and evolution. Indeed, a major subset of SMZL, roughly 30% of the entire cohort, is defined by the expression of IG receptors with heavy variable domains (VH) utilizing the IGHV1-2*04 gene. These VH domains exhibit low-level, yet non-randomly targeted somatic hypermutation (SHM), carry long and often positively charged heavy complementarity-determining region 3 with restricted motifs, and also show biased associations with certain light IG genes, namely IGKV3-20, IGKV1-8 and IGLV2-14. We recently documented that the great majority of SMZL, especially IGHV1-2*04 cases, exhibit intraclonal diversification (ID) in IGHV genes, reflecting ongoing interactions with antigen(s). In this study we further extend the analysis of ID in SMZL focusing on IG light genes. To this end, we performed a comprehensive subcloning analysis of IGKV-IGKJ and IGLV-IGLJ gene rearrangements from 11 SMZL cases; two patients were studied at two different time-points. A total of 311 subcloned sequences (10-38/sample, median, 25) were obtained from 9 IGKV-IGKJ and 4 IGLV-IGLJ rearrangements. Multiple alignment of the subcloned sequences revealed that: (1) only one of 13 studied samples (7.8%) carried identical subclones (no ID); (2) 6/13 (46.1%) carried only unconfirmed mutations (UCMs, mutations in single subclones; unconfirmed ID); and, (3) 6/13 (46.1%) carried confirmed mutations (CMs, identical mutations in at least 2 subclones; confirmed ID). Both IGHV1-2*04 cases of the present series belonged to the confirmed ID category and both displayed intense ID with extensive subclone formation. Among cases positive for ID, the number of nucleotide substitutions introduced by ongoing SHM ranged from 1-21. A total of 66 unique substitutions were identified in 42 positions of the variable domain; 44 of these resulted in the replacement of the germline-encoded amino acid (R), while the remaining 22 were silent (S). The distribution of replacement and silent CMs and UCMs in CDRs and FRs was compatible with a canonical SHM process in that R/S ratios were higher in CDRs, except CDR2. In general, ID was more pronounced in IGKV-IGKJ versus IGLV-IGLJ rearrangements, regardless of the overall mutational load of each rearrangement. The analysis of the same rearrangement at different time-points revealed a consistent ID profile: one case carried only UCMs at both time-points, while the other (IGHV1-2*04 case) exhibited a dynamic pattern of appearing and disappearing CMs. Overall, the presence of mutations in the context of ID was independent of the mutational status, as ID was observed even in minimally mutated cases (98-99.9% germline identity). Moreover, in 3/6 cases with confirmed ID, subclones were classified in ≥2 main mutational groups, indicating early “branching” of the clonal population in subpopulations with distinct mutational profiles. In conclusion, the present study complements the immunogenetic profile of SMZL, offering novel molecular evidence for crosstalk with the microenvironment mediated through the clonotypic B-cell receptors. Furthermore, it underscores the significance of IG light chains in the immune pathogenesis of SMZL. Disclosures: No relevant conflicts of interest to declare.

Distinctive Patterns of Intraclonal Diversification In IGHV1-2*04 Immunoglobulin Receptors of Patients with Splenic Marginal Zone Lymphoma: A of Ongoing Interactions with Antigen?

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2638-2638
Author(s):  
Vasilis Bikos ◽  
Evangelia Stalika ◽  
Nikos Darzentas ◽  
Maria Karypidou ◽  
Panagiotis Baliakas ◽  
...  
Keyword(s):  
Amino Acid ◽  
Research Funding ◽  
Large Scale ◽  
Marginal Zone ◽  
Somatic Hypermutation ◽  
Marginal Zone Lymphoma ◽  
Splenic Marginal Zone Lymphoma ◽  
Antigen Binding Site ◽  
Molecular Features ◽  
Major Subset

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.

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