Biased immunoglobulin heavy variable (IGHV) gene usage and distinctive B-cell receptor (BCR) features have been reported in chronic lymphocytic leukaemia (CLL), which may reflect clonal selection by antigens during disease development. Furthermore, the IGHV gene mutation status distinguishes two clinical entities of CLL, where patients with unmutated IGHV genes have an inferior prognosis compared to those with mutated IGHV genes. Recently, one subgroup of CLL patients expressing the IGHV3-21 gene was found to display highly similar immunoglobulin (IG) gene features, even within the heavy chain complementarity-determining region 3 (HCDR3). Patients in this subgroup typically had a poor prognosis. In paper I, we aimed to identify further subgroups with restricted BCR features among 346 CLL cases. Six subsets were defined which carried virtually identical BCRs in terms of rearranged heavy and light chain (LC) IG genes and CDR3 length and composition. In paper II, we investigated 90 IGHV3-21 cases from diverse geographical locations. We confirmed the highly restricted HCDR3 characteristics in 56% of patients and a biased usage of the IGLV3-21 gene in 72% of cases. Survival analysis also confirmed the poor outcome of this group, irrespective of IGHV gene mutation status and geographical origin. Papers III and IV involved a large-scale analysis of IGH and IG kappa and lambda (IGK/L) gene rearrangements, to define subsets with ‘stereotyped’ BCRs and also to systematically examine the somatic hypermutation (SHM) features of the IG genes in CLL. We studied a cohort of 1967 IGH and 891 IGK/L gene sequences from 1939 patients from 6 European institutions. Over 5300 IGH and ~4700 IGK/L sequences from non-CLL B cells were used as a control data set. In total, 110 CLL stereotyped subsets were defined according to HCDR3 homology. Striking IGK/L gene biases were also evident within subsets, along with distinctive K/LCDR3 features, such as length and amino acid composition. At cohort level, the patterns of mutation appeared to be consistent with that of a canonical SHM mechanism. However, at a subgroup level, certain stereotyped subsets, e.g. IGHV3-21/IGLV3-21 and IGHV4-34/IGKV2-30 CLL, deviated from this pattern. Furthermore, recurrent ‘stereotyped’ mutations occurred in cases belonging to subsets with restricted HCDR3s, in both IGHV and IGK/LV genes, which were subset- and CLL-biased when compared to non-CLL B cells. In conclusion, our findings implicate antigen selection as a significant factor in the pathogenesis of CLL, particularly in cases carrying stereotyped BCRs. The presence of stereotyped mutations throughout the VH and VL domain also indicates involvement of IG regions other than the CDR3 in antigen recognition. Finally, biased IGK/L gene usage and specific K/LCDR3 features are strong indications that LCs are crucial in shaping the specificity of leukemic BCRs, in association with defined heavy chains.

<p>B cell lymphoma (BCL) comprises a biologically and clinically heterogeneous group of tumors deriving from different stages of B cell development. The immunoglobulin (Ig) variable heavy chain (V_H) gene rearrangement is unique for each BCL and can be used to reveal cellular origin, to study signs of antigen selection and to quantify tumor cell load.</p><p>The normal counterpart of mantle cell lymphoma (MCL) has been postulated to be a naïve B cell and in hairy cell leukemia (HCL) it is considered to be a post-germinal centre B cell. We analyzed the V_H gene rearrangements in 110 MCLs and 32 HCLs by PCR amplification and sequencing. Most MCLs (84%) displayed V_H genes lacking somatic hypermutation (SHM), thus correlating to a naïve cell origin, whereas a subgroup (16%) showed SHM, implying derivation from a more differentiated B cell. In HCL, a majority of cases (84%) displayed SHM with signs of intraclonal heterogeneity and 16% had unmutated V_H genes, thus questioning the cell of origin in HCL. Biased usage of particular V_H genes was detected in both HCL (V_H3-30) and MCL (V_H3-21 and V_H4-34), which indicates that antigen selection may be involved in lymphoma development. Furthermore, V_H3-21⁺ MCLs showed a highly restricted V_λ3-19 gene use and they also had a superior outcome compared to other MCLs.</p><p>Rearrangement analysis of 67 V_H3-21⁺ chronic lymphocytic leukemia (CLL) cases from three different countries verified, regardless of geographical origin, the short and highly homologous complementarity determining region 3s and the strikingly biased usage of the V_λ2-14 gene (75%), as previously reported in CLL. This further supports that antigen selection by a common antigenic epitope may have occurred in V_H3-21⁺ CLLs. </p><p>In an autologous transplantation study of 30 multiple myeloma patients, we quantified the tumor content in the autografts before and after stem cell selection using clone-specific PCR. We conclude that stem cell selection reduced the number of clonal cells linearly, but purging could not totally eliminate the tumor cells from the graft, thus increasing the risk of a relapse.</p><p>Altogether, our data allowed us to define new BCL subsets and to gain insights into the potential role of antigen selection in BCL development as well as the monitoring of tumor cell load using Ig gene rearrangements analysis. </p>

B cell lymphoma (BCL) comprises a biologically and clinically heterogeneous group of tumors deriving from different stages of B cell development. The immunoglobulin (Ig) variable heavy chain (VH) gene rearrangement is unique for each BCL and can be used to reveal cellular origin, to study signs of antigen selection and to quantify tumor cell load. The normal counterpart of mantle cell lymphoma (MCL) has been postulated to be a naïve B cell and in hairy cell leukemia (HCL) it is considered to be a post-germinal centre B cell. We analyzed the VH gene rearrangements in 110 MCLs and 32 HCLs by PCR amplification and sequencing. Most MCLs (84%) displayed VH genes lacking somatic hypermutation (SHM), thus correlating to a naïve cell origin, whereas a subgroup (16%) showed SHM, implying derivation from a more differentiated B cell. In HCL, a majority of cases (84%) displayed SHM with signs of intraclonal heterogeneity and 16% had unmutated VH genes, thus questioning the cell of origin in HCL. Biased usage of particular VH genes was detected in both HCL (VH3-30) and MCL (VH3-21 and VH4-34), which indicates that antigen selection may be involved in lymphoma development. Furthermore, VH3-21+ MCLs showed a highly restricted Vλ3-19 gene use and they also had a superior outcome compared to other MCLs. Rearrangement analysis of 67 VH3-21+ chronic lymphocytic leukemia (CLL) cases from three different countries verified, regardless of geographical origin, the short and highly homologous complementarity determining region 3s and the strikingly biased usage of the Vλ2-14 gene (75%), as previously reported in CLL. This further supports that antigen selection by a common antigenic epitope may have occurred in VH3-21+ CLLs. In an autologous transplantation study of 30 multiple myeloma patients, we quantified the tumor content in the autografts before and after stem cell selection using clone-specific PCR. We conclude that stem cell selection reduced the number of clonal cells linearly, but purging could not totally eliminate the tumor cells from the graft, thus increasing the risk of a relapse. Altogether, our data allowed us to define new BCL subsets and to gain insights into the potential role of antigen selection in BCL development as well as the monitoring of tumor cell load using Ig gene rearrangements analysis.

<p>B cell lymphomas and leukemias are heterogeneous tumors with different cellular origins. Analysis of immunoglobulin (Ig) genes enables insight into the B cell progenitor, as Ig somatic hypermutation correlates with antigen-related B cell transit through the germinal center (GC). Also, restricted Ig variable heavy chain (V_H) gene repertoires in B cell malignancies could imply antigen selection during tumorigenesis. The length of telomeres has been shown to differ between GC B cells and pre/post-GC B cells, possibly representing an alternative angle to investigate B cell tumor origin. </p><p>Mantle cell lymphoma (MCL), previously postulated to derive from a naïve, pre-GC B cell, was shown to have an Ig-mutated subset (18/110 MCLs, 16%), suggestive of divergent cellular origin and GC exposure. Another subset of MCL (16/110, 15%), characterized by V_H3-21/V_λ3-19 gene usage, alludes to a role for antigen(s) in pathogenesis, also possible for hairy cell leukemia (HCL) in which the V_H3-30 gene (6/32, 19%) was overused. HCL consisted mainly of Ig-mutated cases (27/32, 84%) with low level intraclonal heterogeneity, contrasting with the proposed post-GC origin, for both Ig-mutated and Ig-unmutated HCLs. For MCL and HCL, derivation from naïve or memory marginal zone B cells which may acquire mutations without GC transit are tempting speculations, but currently little is known about this alternative immunological pathway. Heavily mutated Ig genes without intraclonal heterogeneity were demonstrated in lymphoplasmacytic lymphoma/Waldenström’s macroglobulinemia (13/14, 93%), confirming that the precursor cell was transformed after GC affinity maturation. Telomere length analysis within 304 B cell tumors revealed variable lengths; shortest in the Ig-unmutated subset of chronic lymphocytic leukemia, longest in the GC-like subtype of diffuse large B cell lymphoma, and homogeneous in MCL regardless of Ig mutation status. However, telomere length is complex with regard to GC-related origin.</p><p>In summary, this thesis has provided grounds for speculation that antigens play a role in MCL and HCL pathogenesis, although the potential antigens involved are currently unknown. It has also enabled a more informed postulation about the cellular origin of B cell tumors, which will ultimately enhance understanding of the biological background of the diseases. </p>

B cell lymphomas and leukemias are heterogeneous tumors with different cellular origins. Analysis of immunoglobulin (Ig) genes enables insight into the B cell progenitor, as Ig somatic hypermutation correlates with antigen-related B cell transit through the germinal center (GC). Also, restricted Ig variable heavy chain (VH) gene repertoires in B cell malignancies could imply antigen selection during tumorigenesis. The length of telomeres has been shown to differ between GC B cells and pre/post-GC B cells, possibly representing an alternative angle to investigate B cell tumor origin. Mantle cell lymphoma (MCL), previously postulated to derive from a naïve, pre-GC B cell, was shown to have an Ig-mutated subset (18/110 MCLs, 16%), suggestive of divergent cellular origin and GC exposure. Another subset of MCL (16/110, 15%), characterized by VH3-21/Vλ3-19 gene usage, alludes to a role for antigen(s) in pathogenesis, also possible for hairy cell leukemia (HCL) in which the VH3-30 gene (6/32, 19%) was overused. HCL consisted mainly of Ig-mutated cases (27/32, 84%) with low level intraclonal heterogeneity, contrasting with the proposed post-GC origin, for both Ig-mutated and Ig-unmutated HCLs. For MCL and HCL, derivation from naïve or memory marginal zone B cells which may acquire mutations without GC transit are tempting speculations, but currently little is known about this alternative immunological pathway. Heavily mutated Ig genes without intraclonal heterogeneity were demonstrated in lymphoplasmacytic lymphoma/Waldenström’s macroglobulinemia (13/14, 93%), confirming that the precursor cell was transformed after GC affinity maturation. Telomere length analysis within 304 B cell tumors revealed variable lengths; shortest in the Ig-unmutated subset of chronic lymphocytic leukemia, longest in the GC-like subtype of diffuse large B cell lymphoma, and homogeneous in MCL regardless of Ig mutation status. However, telomere length is complex with regard to GC-related origin. In summary, this thesis has provided grounds for speculation that antigens play a role in MCL and HCL pathogenesis, although the potential antigens involved are currently unknown. It has also enabled a more informed postulation about the cellular origin of B cell tumors, which will ultimately enhance understanding of the biological background of the diseases.

Les Lymphomes Primitifs Vitréo-Rétiniens (LPVR) représentent un sous-type de Lymphome Primitif du Système Nerveux Central (LPSNC). Ces hémopathies très rares sont caractérisées par leur localisation anatomique atypique, dans des sites physiologiquement dépourvus de lymphocytes B. Les lymphomes du SNC sont rattachés histologiquement aux Lymphomes B Diffus à Grandes Cellules (LBDGC) de type post-germinatif (ABC). L’objectif de notre étude était de définir le répertoire immunologique (chaînes lourdes et légères) des LPVR et des LPSNC, et de les comparer aux LBDGC. Nous avons mené une étude immunologique détaillée de ces tumeurs afin de rechercher des éléments de réponse expliquant ces localisations ectopiques. Notre projet, réalisé sur la plus grande série de LPVR à ce jour, a mis en évidence un biais de répertoire majeur, avec une sur-représentation massive du gène IGHV4-34 (63,6% des cas), significativement plus utilisé dans les LPVR comparativement aux LPSNC et aux LBDGC systémiques. Bien que la proportion de ce gène soit élevée dans d’autres SLP, cette fréquence n’a jamais été atteinte. Un subset a été décrit pour 50% des LPVR utilisant le gène IGHV3-7. Ces données suggèrent fortement l’implication d’un antigène dans leur développement. En conclusion, le LPVR représente un modèle surprenant et singulier de lymphome dirigé par l’antigène, dont l’identification offrirait des perspectives physiopathologiques et thérapeutiques prometteuses. / Primary vitroretinal lymphoma (PVRL) is a high-grade lymphoma considered as a subtype of primary central nervous system lymphoma (PCNSL). Unusual localization is a feature of these rare entities. The vast majority of cases are diffuse large B cell lymphoma (DLBCL), mostly of activated B-cell (ABC). To investigate whether PVRLs display a specific IG repertoire contributing to explain their unusual localization, we analysed in detail the IG heavy and light chain sequences from PVRL and PCNSL cases, and we compared their repertoire to that of a publicly available IG heavy chain sequences dataset from systemic ABC-type DLBCLs. Our study was carried out on the largest PVRL series reported to date and showed that PVRL displayed a strikingly biased repertoire as the IGHV4-34 gene was used in 63.6% of cases. The frequency was significantly higher in PVRL compared to PCNSL and DLBCL. This gene has been repeatedly found to be preferentially used in various B-cell malignancies, but never to such an extent. Half of PVRL cases expressing the IGHV3-7 gene had stereotyped VH CDR3 features (subset). Altogether our data showed that PVRLs display a very biased IG repertoire strongly suggesting that antigen selection plays a major role in their development. Thus, PVRL display a highly restricted IG repertoire indicative of antigen selection, and distinct from that of PCNSL. Antigen(s) identification may provide promising perspectives in physiopathology concepts and therapeutic approaches.