Emanuela Carlotti

Transformation of follicular lymphoma to diffuse large B-cell lymphoma may occur by divergent evolution from a common progenitor cell or by direct evolution from the follicular lymphoma clone

To investigate the cell of origin linking follicular (FL) and transformed (t-FL) lymphomas, we analyzed the somatic hypermutation (SHM) pattern of the variable region of the immunoglobulin heavy gene (IgH-VH) in 18 sequential FL/t-FL samples and a father (donor) and son (recipient), who developed FL and t-FL, after transplantation. Genealogic trees showed a pattern compatible with a common progenitor cell (CPC) origin in 13 cases. The identification of the t-FL clonotype in the previous FL sample and of the putative CPC sequence in both the FL/t-FL biopsies showed that the intraclonal diversity of FL and t-FL germinal centers (GCs) is more intricate than previously described, and all 3 clonotypes (CPC, FL, t-FL) may occur simultaneously within the same lymph node. On the basis of the father/son model, this CPC must be long-lived, providing a possible explanation for the incurable nature of this disease.

The presence of TP53 mutation at diagnosis of Follicular Lymphoma identifies a high-risk group of patients with shortened time to disease progression and poorer overall survival

The International Prognostic Index and the Follicular Lymphoma International Prognostic Index are widely used for the risk assessment of follicular lymphoma (FL). Although molecular studies have provided insight into the biology of FL, no molecular marker has impacted on treatment stratification. Because TP53 mutations are associated with poor prognosis in hematologic malignancies, we investigated the prognostic value of TP53 mutation at diagnosis in FL. Heterozygous TP53 mutation was detected in 12 of 185 (6%) analyzed cases. Mutation was associated with older age (P = .02) and higher International Prognostic Index score (P = .04). On multivariate analysis, TP53 mutation correlated with shorter progression-free survival (P < .001) and overall survival (P = .009). TP53 mutation was associated with low expression of the immune-response 1 gene expression signature (P = .016) and with an unfavorable gene expression-based survival predictor score (P < .001), demonstrating for the first time that molecular features of the malignant cell may correlate with the nature of the immune response in FL.

Genome-wide detection of recurring sites of uniparental disomy in follicular and transformed follicular lymphoma

Single-nucleotide polymorphism (SNP) array analysis was performed using the 10K GeneChip array on a series of 26 paired follicular lymphoma (FL) and transformed-FL (t-FL) biopsies and the lymphoma cell lines SCI-1, DoHH2 and RL2261. Regions of acquired homozygosity were detected in 43/52 (83%) primary specimens with a mean of 1.7 and 3.0 aberrations in the FL and t-FL, respectively. A notable feature was the occurrence of recurring sites of acquired uniparental disomy (aUDP) on 6p, 9p, 12q and 17p in cell lines and primary samples. Homozygosity of 9p and 17p arose predominantly in t-FL and in three cases rendered the cell homozygous for a pre-existing mutation of either CDKN2A or TP53. These data suggest that mutation precedes mitotic recombination, which leads to the removal of the remaining wild-type allele. In all, 18 cases exhibited abnormalities in both FL and t-FL samples. In 10 cases blocks of homozygosity were detected in FL that were absent in the subsequent t-FL sample. These differences support the notion that FL and t-FL may arise in a proportion of patients by divergence from a common malignant ancestor cell rather than by clonal evolution from an antecedent FL.

Single cell cloning and recombinant monoclonal antibodies generation from RA synovial B cells reveal frequent targeting of citrullinated histones of NETs.

Objectives Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated antigens with generation of anti-citrullinated peptide/proteins antibodies (ACPA). Currently, the nature and source of citrullinated antigens driving the humoral autoimmune response within synovial ectopic lymphoid structures (ELS) is a crucial unknown aspect of RA pathogenesis. Here we characterised the autoreactive B-cell response of lesional B cells isolated from ELS+RA synovium. Methods Single synovial tissue CD19+cells were Fluorescence Activated Cell Sorting (FACS)-sorted and VH/VL Ig genes cloned to generate recombinant monoclonal antibodies (rmAbs) from patients with ELS+/ACPA+RA. Results RA-rmAbs immunoreactivity analysis provided the following key findings: (1) in a chIP-based array containing 300 autoantigens and in a ‘citrullinome’ multiplex assay, a strong reactivity against citrullinated histones H2A/H2B (citH2A/H2B) was observed in ∼40% of RA-rmAbs, followed by cit-fibrinogen and cit-vimentin; (2) anti-citH2A/H2B-reactive RA-rmAbs (but not anti-citH2A/H2B negative) selectively recognised neutrophil extracellular traps (NETs) from peripheral blood and/or RA joint neutrophils; (3) anti-citH2A/citH2B and anti-NET immunobinding was dependent on affinity maturation and was completely abrogated following reversion of hypermutated IgVH/VL genes to germline sequences; (4) ELS+ (not ELS−) RA synovial tissues engrafted into Severe Combined ImmunoDeficiency (SCID) mice released human anti-citH2A/citH2B and anti-NET antibodies in association with the intra-graft expression of CXCL13 and lymphotoxin (LT)-β, two master regulators of ELS. Conclusion We provided novel evidence that B cells differentiated within synovial ELS in the RA joints frequent target deiminated proteins which could be generated during NETosis of RA synovial neutrophils including histones. Thus, NETs could represent a source of citrullinated antigens fuelling the ACPA autoimmune response within the RA synovium.

Stage I/II follicular lymphoma: spread of bcl‐2/IgH+ cells in blood and bone marrow from primary site of disease and possibility of clearance after involved field radiotherapy

Stage I/IIA follicular lymphoma (FL) is considered a localised disease that can be adequately treated with radiotherapy alone. Bone marrow (BM) and peripheral blood (PB) involvement in FL was investigated by polymerase chain reaction (PCR) in a series of 24 consecutive patients with histologically revised diagnosis and treated with involved field radiotherapy. Despite the limited stage, Bcl‐2/IgH+ cells were found at diagnosis in PB and/or BM of 16 patients (66·6%). After treatment, in 9/15 Bcl‐2/IgH positive evaluable patients, a disappearance of Bcl‐2/IgH+ cells was observed, which persisted after a median follow‐up of 43·5 months (range 11–70) in all but one patient. Quantitative PCR demonstrated the feasibility of clearing PB and BM Bcl‐2+ cells after local irradiation of the primary site of the disease only when the basal number of lymphoma cells was <1:100 000. Patients with Bcl‐2/IgH+ cells at diagnosis or after treatment had a higher likelihood of relapse. Thus, despite a negative BM biopsy, the majority of localised FL Bcl‐2/IgH+ cells were found in the PB and BM. Lymphoma cells can reversibly spread from the affected lymph node to PB and BM and, in a proportion of cases, durably disappear after irradiation. The possibility of a persistent lymphoma cell clearance is proportional to the amount of cells detected at presentation by quantitative PCR.

SNP rs6457327 in the HLA region on chromosome 6p is predictive of the transformation of follicular lymphoma

Inherited risk determinants for follicular lymphoma (FL) have recently been described in the immune gene-rich human leukocyte antigen region on chromosome 6p. The known importance of host immune response to FL survival led us to evaluate these germline factors in FL outcome. We confirm the association of single nucleotide polymorphisms rs10484561 (P = 3.5 × 10⁻⁹) and rs6457327 (P = .008) with risk of FL and demonstrate that rs6457327 predicts both time to (P = .02) and risk of (P < .01) FL transformation independently of clinical variables, including the Follicular Lymphoma International Prognostic Index.

Molecular characterization of a new recombination of the SIL/TAL-1 locus in a child with T-cell acute lymphoblastic leukaemia

Summary. Deletions involving the SIL‐TAL‐1 locus are seen in 15% of T‐acute lymphoblastic leukaemias (T‐ALL). To date, seven deletions have been described, spreading over 90 kb of chromosome 1, fusing SIL to the TAL‐1 gene and resulting in over expression of TAL‐1. During the diagnostic screening of the TAL‐1 deletion in 176 T‐ALL patients, we identified one case showing a new SIL rearrangement. A novel fusion transcript was identified between the SIL exon 1a and an unknown sequence (633‐cDNA). Polymerase chain reaction (PCR) screening of a human cDNA library confirmed the existence of this transcript. Using long‐distance PCR on patient DNA, we obtained a genomic fragment containing SIL exon 1b, a portion of intron 1b, an unknown sequence and the 633 sequence. Using DNA from healthy donors, a partial genomic map of 633‐DNA was found to be identical to the restriction map of the PCR fragment amplified from patient DNA. To define the chromosomal origin of 633‐DNA, a YAC human genomic library was screened. Two clones containing 633‐DNA were found, mapping to chromosomal region 1p32 and both contained SIL and TAL‐1 sequences. By searching GenBank, we identified PAC RP1‐18D14 which contains SIL, TAL‐1 and 633‐DNA, confirming this novel rearrangement as a new deletion of the SIL/TAL‐1 locus.

High Throughput Sequencing Analysis of the Immunoglobulin Heavy Chain Gene from Flow-Sorted B Cell Sub-Populations Define the Dynamics of Follicular Lymphoma Clonal Evolution

Understanding the dynamics of evolution of Follicular Lymphoma (FL) clones during disease progression is important for monitoring and targeting this tumor effectively. Genetic profiling of serial FL biopsies and examples of FL transmission following bone marrow transplant suggest that this disease may evolve by divergent evolution from a common ancestor cell. However where this ancestor cell resides and how it evolves is still unclear. The analysis of the pattern of somatic hypermutation of the immunoglobulin gene (Ig) is traditionally used for tracking the physiological clonal evolution of B cells within the germinal center and allows to discriminate those cells that have just entered the germinal center and display features of ancestor cells from those B cells that keep re-circulating across different lymphoid organs. Here we investigated the pattern of somatic hypermutation of the heavy chain of the immunoglobulin gene (IgH-VH) in 4 flow-sorted B cells subpopulations belonging to different stages of differentiation, from sequential lymph node biopsies of cases displaying diverse patterns of evolution, using the GS-FLX Titanium sequencing platform. We observed an unexpectedly high level of clonality, with hundreds of distinct tumor subclones in the different subpopulations from the same sample, the majority detected at a frequency <10−2. By using a lineage trees analysis we observed in all our FL and t-FL cases that the oligoclonal FL population was trapped in a narrow intermediate stage of maturation that maintains the capacity to undergo SHM, but was unable to further differentiate. The presence of such a complex architecture highlights challenges currently encountered in finding a cure for this disease.

Clinical relevance of MDM2 SNP 309 and TP53 Arg72Pro in follicular lymphoma

Tumor protein 53 (TP53) is critical to cell cycle control and is the most common mutational target in germinal center lymphomas. However, these mutations occur infrequently at diagnosis (<10%) in follicular lymphoma (FL), and are more commonly associated with disease progression or transformation to

A1.31 Monoclonal antibodies from CD19+ synovial B cells of RA patients with tertiary lymphoid structures display a strong immunoreactivity towards citrullinated histones from neutrophils NETs

Background and Objectives Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated proteins. Moreover, RA patients are characterised by increased neutrophils in their synovial fluid, in particular at early stage of the disease. Recent evidence suggests a critical role of neutrophils in sustaining the inflammatory response in the RA joint. Around 40% of patients display synovial tertiary lymphoid structures (TLS) with functional B cell follicles supporting a germinal-centre response and local autoantibody production. However, the nature of the main (auto)antigenic reactivity of synovial B cells is unknown. Here we characterised the autoreactive B cell response of lesional B cells isolated from TLS + RA synovium. Materials and Methods Single CD19+ B cells were FACS sorted from synovial cell suspension of 4 TLS + RA patients. RNA was used to amplify Ig VH and VL genes and PCR products were cloned and expressed as recombinant monoclonal antibodies displaying identical specificity of the original B cells. Recombinant antibodies were then tested 1) to determine the frequency of polyreactive clones and 2) to define their immunoreactivity towards native and citrullinated antigens using a synovial antigen microarray platform. Results We obtained 139 individual VH sequences of which 33% were IgM, 40% IgG, 27% IgA and 175 VL sequences and we generated a total of 66 complete (H + L chains) recombinant monoclonal antibodies. Analysis of the VH gene somatic mutation rate showed evidence of antigen selection and intra-synovial clonal diversification. No skewed distribution of the VH and VL gene usage was observed. Around 30% of synovial monoclonal antibodies were reactive towards citrullinated histones in the antigen microarray, in particular citH2A and citH2B. This reactivity was confirmed by citH2A and citH2B ELISA. Moreover, when the synovial monoclonal antibodies were tested on neutrophil extracellular traps (NETs), which contained citrullinated histones, reactivity towards NETs proteins but not neutrophil nuclear antigens was observed. Conclusions Here we provided novel evidence that highly mutated, locally differentiated B cells within RA synovial germinal centre-like structures display a strong immunoreactive bias towards citrullinated histones which likely derived from neutrophil NETs that continuously form in the RA synovial fluid. This suggests that citrullinated histones are the main antigens driving in situ B cell activation and differentiation sustaining the humoral autoimmune response within the RA joints.