ALBERT

Description: The IGHV4-34 gene is very frequent (~10%) in the B cell receptor immunoglobulin (BcR IG) gene repertoire of chronic lymphocytic leukemia (CLL). Over 30% of IGHV4-34 CLL cases can be assigned to different subsets with stereotyped BcR IG. The largest is subset #4 which represents ~1% of all CLL and ~10% of IGHV4-34 CLL and is considered a prototype for indolent disease. The BcR IG of a great majority (~85%) of IGHV4-34 CLL cases carry a significant load of somatic hypermutation (SHM), often with distinctive SHM patterns. This holds especially true for stereotyped subsets and is suggestive of particular modes of interactions with the selecting antigen(s). In detail, subsets #4 and #16, both involving IgG-switched cases (IgG-CLL), exhibit the greatest sequence similarity in SHM profiles, whereas they differ in this respect from IgM/D subsets #29 and #201. Prompted by these observations, here we explored the extent that these subset-biased SHM profiles in different IGHV4-34 stereotyped subsets were reflected in distinct demographics, clinical presentation, genomic aberrations and outcomes. Within a multi-institutional series of 20,331 CLL patients, 1790 (8.8%) expressed IGHV4-34 BcR IG. Following established bioinformatics approaches for the identification of BcR IG stereotypy, 573/1790 IGHV4-34 CLL cases (32%) were assigned to stereotyped subsets; of these, 340 cases (19% of all IGHV4-34 CLL and 60% of stereotyped IGHV4-34 cases) belonged to subsets #4, #16, #29 and #201, all concerning IGHV-mutated CLL (M-CLL). Clinicobiological information was available for 275/340 patients: #4, n=150; #16, n=44; #29, n=39; and #201, n=42. Comparisons between subsets revealed no differences in gender and age distribution. Interestingly, however, 36-43% of each subset cases were young for CLL (defined as patients aged ≤55 years), which is higher compared to general CLL cohorts, where young patients generally account for ~25% of cases. In contrast, significant differences were identified between subsets regarding: (i) disease stage at diagnosis, with 〉90% of IgG subsets #4 and #16 diagnosed at Binet stage A versus 83% in subset #201 and 74% in subset #29 (p=0.029); (ii) CD38 expression, ranging from 1% in subset #4 to 10% in subset #201 (p=0.013); (iii) the distribution of del(13q), peaking at a remarkable 92% in subset #29 versus only 37% in subset #16 (p

Description: In MM patients relapsing after MRD-negativity, the disease could reemerge from immature cells or from undetectable MRD. However, it remains unknown if immature cells have the same genetic background as MM plasma cells (PCs), as well as the amount of MRD that persists below the limit of detection (LOD) of next-generation techniques. To obtain further insight, we compared the biological landscape of MM PCs at diagnosis to that of CD34 progenitors, B cells and normal PCs isolated from patients with negative MRD by next-generation flow (NGF) after treatment. We performed whole-exome sequencing (WES, mean depth: 90x) with the 10XGenomics Exome Solution for low DNA-input as well as deep NGS of B-cell receptor immunoglobulin (BcR IG) gene rearrangements (mean, 69,975 sequences), in a total of 68 cell-samples isolated from the bone marrow (BM) of 7 MM patients with MRD-negativity by EuroFlow NGF after induction with VRD and auto-transplant (GEM2012MENOS65 trial). Patients with negative MRD were intentionally selected to avoid contamination with MM PCs during sorting of CD34 progenitors, B-cell precursors, mature B cells and normal PCs after induction and transplant. We investigated in these populations the presence of somatic mutations and clonotypic BcR Ig rearrangements detectable in MM PCs sorted at diagnosis, using peripheral blood T cells as germline control. We also performed WES in matched diagnostic MM PCs and MRD cells persisting after VRD induction in 14 cases as control. In another 6 patients with untreated MM, we performed single-cell RNA and BcR IG sequencing (scRNA/BcRIGseq) of total BM B cells and PCs (n=16,380) to investigate before treatment, if the clonotypic BcR IG sequence of MM PCs was detectable in other B cell stages defined by their molecular phenotype. We used multidimensional flow cytometry (MFC) to investigate the frequency of B cell clonality in BM samples from a larger series of 195 newly-diagnosed MM patients, prospectively enrolled in the GEM-CLARIDEX trial. Somatic mutations present in diagnostic MM PCs were detectable in the lymphopoiesis of 5/7 patients achieving MRD-negativity after treatment. In one case, out of 55 mutations present in diagnostic MM PCs, a single mutation in PCSK1N (VAF: 0.30) was detectable in normal PCs. In the other four patients, a total of 85 mutations were present in MM PCs and up to 10 (median VAF, 0.16) were found all the way from CD34 progenitors into B-cell precursors, mature B cells and normal PCs, but not in T cells. Of note, most mutations were reproducibly detected in each cell type after induction and after transplant. All somatic mutations shared by MM PCs and normal cells were non-recurrent, and genes recurrently mutated in MM (eg. ACTG1, ATM, DIS3, FAM46C, KRAS, LTB, MAX, TRAF3) were found in MM PCs but never in normal cells. Copy number alterations (CNA) were found only in MM PCs. By contrast, up to 513/827 (62%) mutations and 48/67 (72%) CNA were detectable in matched diagnostic MM PCs and persistent MRD cells, indicating that the few somatic variants present in normal cells were unlikely related to contaminating MRD below NGF's LOD. Accordingly, MM clonotypic BcR IG rearrangements were detectable in normal PCs (4/7patients) and in immature B cells (5/7 patients) but at much lower frequencies (mean of 0.02% in both). Of note, 9 additional clonotypes (mean 8.4%) were found in MM PCs of 5/7 patients (range, 1-3). scRNR/BcRIGseq unveiled that clonotypic cells were confined mostly but not entirely within PC clusters, and that in 1 patient another clonotype was detectable in mature B cells. Accordingly, using MFC we found in a larger series that 25/195 (13%) of newly-diagnosed MM patients display B-cell clonality (median of 0.7% BM clonal B cells, range 0.02%-6.3%). In conclusion, we show for the first time that MM patients bear somatic mutations in CD34 progenitors that specifically differentiate into the B cell lineage, likely before the disease onset. Because diagnostic, MRD (and relapse) MM PCs display great genetic similarity, these results suggest that undetectable MRD

Description: Immunoglobulin (IG) G-switched chronic lymphocytic leukemia (G-CLL) is highly enriched for 3 stereotyped CLL subsets, utilizing either the IGHV4-34 gene, namely mutated subsets #4 and #16, or the IGHV4-39 gene, namely unmutated subset #8. These subsets, collectively accounting for ~30% of all G-CLL, are not represented within the common IgM/D variant, thus prompting speculations about distinct ontogenetic origin and/or immune triggering, as well as raising questions regarding the timing of class-switch recombination (CSR) in relation to malignant transformation. Considering the above, we sought to investigate the potential existence of B cells expressing clonotypic mu transcripts within the bulk of IgG-switched CLL cells in cases assigned to the aforementioned subsets. Using high-throughput next-generation sequencing (NGS, MiSeq Illumina), we interrogated the IgM+ B-cell repertoire of CLL subset #4 (n=8), subset #16 (n=1) and subset #8 (n=2) for the presence of clonotypic mu transcripts. PCR amplicons were generated from cDNA using a set of IGHV4/IGHM primers. The paired-end Illumina protocol allowed sequencing of the complementarity determining region 3 (CDR3) twice/read, thus increasing the accuracy of results. For 3/8 subset #4 cases multiple blood samples of the same time point were analyzed as reproducibility controls. A purpose-built bioinformatics algorithm was developed for raw NGS data processing, which included: (i) quality filtering of reads; (ii) merging of paired-end reads via local alignment; (iii) preparation of filtered-in fasta sequences for submission to the IMGT/HighV-QUEST tool; and, (iv) IMGT/HighV-QUEST metadata mining for subset-specific B-cell receptor (BcR) IG rearrangements. Subset-specific CDR3 motifs were defined according to established criteria. Overall, 7,125,958 IGHV-IGHD-IGHJ-IGHC rearrangements (189,988-673,835/sample) were included in the search for stereotyped motifs, corresponding to 1,056,967 distinct clonotypes (i.e. BcR IG rearrangements with a particular IGHV gene and amino acid CDR3 sequence) (7,163-123,276/sample, median=76,109). Regarding subset #4, 7/8 cases exhibited mu transcripts of subset #4-specific IG rearrangements ("subset #4 M-clonotypes"); by definition, these rearrangements utilized the IGHV4-34/IGHJ6 genes and had identical CDR3 length (20 amino acids), however their CDR3 amino acid composition varied (2-75 distinct subset #4 M-clonotypes/sample, median=8). In 5/7 cases these subset #4 M-clonotypes were characterized by CDR3s that were identical and/or highly similar (≤2 amino acid differences, ≥ 90% identity) to the CDR3 of the IgG-switched CLL clone. The M-clonotypes expressing CDR3s identical to those of the IgG-switched CLL clone represented the most expanded subset #4 M-clonotype within the sample, while the less expanded, "satellite" clonotypes may represent subclones that were selected against due to lower affinity with the driving antigen. The possibility that these “satellite” clonotypes derive from PCR and/or sequencing error cannot be a priori excluded, however replicate sample analysis produced identical subset #4 M-clonotypes in all cases tested, thus raising confidence in the accuracy of the data. Analysis of the subset #16 case yielded similar results, i.e. 2 subset #16 M-clonotypes, one of which was identical to the IgG-switched clonotypic BcR IG. Both subset #8 cases also carried subset #8 M-clonotypes, yet only one case exhibited an M-clonotype with a CDR3 identical to that of the respective G-CLL clone. Interestingly, this M-clonotype was accompanied by many highly similar, less expanded “satellite” clonotypes (n=109), raising the possibility that SHM may be occurring in (pre-)CLL clones carrying truly unmutated IGHV genes, but pass unnoticed due to negative selection. Although their actual frequency cannot be conclusively determined due to the inherent limitations of PCR-based NGS analysis, subset-specific rearrangements represented a very minor fraction of the sequenced IGHV4/IGHM clonotypes in all cases tested (median frequency 0.04%). Overall, our findings suggest that while CLL clones are primed prior to CSR for malignant transformation on the basis of their BcR IG features, G-CLL quickly transits through CSR either because full-blown malignant transformation occurs at a later time point, or because CSR offers a selective advantage to the malignant clone. Disclosures No relevant conflicts of interest to declare.

Description: Chronic idiopathic neutropenia (CIN) is an acquired disorder of granulopoiesis characterized by prolonged neutropenia, mainly affecting middle-age females of the HLA-DRB1*1302 type. The defective hematopoiesis in CIN can be mainly attributed to accelerated Fas-mediated death of the CD34+/CD33+ granulocytic progenitors, secondary to an inflammatory bone marrow (BM) microenvironment. Crucial to CIN pathogenesis are the increased numbers of activated T cells identified in both peripheral blood (PB) and BM of CIN patients, supporting an immune pathogenesis. Using Sanger sequencing, we previously reported that the T-cell receptor (TR) gene repertoire in CIN is skewed, indicating antigen selection in CIN ontogeny. However, the analytical depth afforded by Sanger sequencing is limited, hindering definitive conclusions. In order to obtain a truly comprehensive view into the role of antigen drive in CIN, using next generation sequencing (NGS) we interrogated the TR repertoire of 13 patients (8 females, 5 males) included in our previous study as well as a healthy female. TRBV-TRBD-TRBJ gene rearrangements were amplified according to the BIOMED2 protocol on either genomic DNA or cDNA isolated from CD8+ cells of PB (n=4) or BM (n=10) samples. PCR products were used as a substrate for paired-end sample preparation (Illumina) and subjected to NGS on the MiSeq Illumina Platform. The raw NGS data were preprocessed with a dedicated pipeline for this purpose, including: (i) quality filtering of each read; (ii) merging of paired-end reads via local alignment; (iii) preparation of fasta files for submission to the IMGT/High V-QUEST tool; and, (iv) IMGT/High V-QUEST metadata analysis, interpretation and visualization. Overall, 6,196,980 TRBV-TRBD-TRBJ gene rearrangements were analyzed (130,020-1,037,680 /case) of which 5,317,609 were productive since they used functional TRBV genes and also carried in-frame CDR3. Rearrangements with identical TRBV gene usage and CDR3 sequence were defined as clonotypes. For repertoire analyses, clonotypes rather than single rearrangement sequences were considered. Overall, 553,145 unique clonotypes were identified (median 39,510; range 7,732-172,253/case), of which 408,744 represented singletons. All clonotypes from the Sanger analysis were detected by NGS as well. Among the 46 functional TRBV genes identified, the most frequent were: TRBV29-1 (13.9%), TRBV19 (6.7%), TRBV12-3 (5.6%), TRBV6-5 (5.4%), TRBV27 (4.9%) and TRBV6-1 (4.0%), collectively accounting for 40,5% of the TRBV repertoire; the TRBV29-1 gene predominated in 9/13 CIN cases. All CIN cases were found to carry distinct expanded clonotypes (median 10,314; range 2,279-40,245/case). The predominant clonotype ranged in frequency from 5.25 to 20.2% of the total clonotypes observed in each case. This contrasts significantly (p

Description: Immunoglobulin heavy chain variable gene (IGHV) replacement or "VH replacement" (VHR) modifies a rearranged IGHV-D-J sequence by replacing the original IGHV gene with another. This process leaves a detectible "footprint" at the IGHV-D junction of the existing sequence. Roughly 33% of chronic lymphocytic leukemia (CLL) cases exhibit stereotyped B cell receptors (BCRs) often characterized by signature VH CDR3 amino acids. Various mechanisms have been put forth to account for stereotypy in CLL. An overarching hypothesis is that the stereotyped BCRs are antigen driven. Within this concept, a variety of mechanisms could lead to the signatures including somatic mutations and addition/deletion of nucleotides at junctional regions. Here we explore the possibility that VHR provides another mechanism to account for some of the stereotyped rearrangements and some of their signature VH CDR3 amino acid residues in CLL. We examined IG sequences of 26,642 CLL cases and ~16 million healthy controls (HC) to find relic footprints as indicators of VHR. This was done using the VHRFA program developed by Lin Huang et al (PLoS ONE, 2013), as well as our own program which duplicates the VHRFA results but is better able to process large numbers of sequences. The frequency of VHR was similar in CLL and HC (11.6 and 11.9%, respectively). Focusing solely on CLL sequences to define a relationship between VHR and stereotypy, we found highly significant differences in VHR frequencies between stereotyped (n=8,568) and non-stereotyped cases (n=18,074), with stereotyped cases exhibiting VHR at a greatly reduced frequency (7.7% vs. 13.5%, respectively). When comparing VHR frequencies between stereotyped cases and non-stereotyped cases that used the same IGHV, we found that the number of subsets with low VHR exceeded those with elevated VHR ~2:1, accounting for the overall VHR in stereotyped cases being lower than non-stereotyped cases. Further restricting comparisons of stereotyped subsets to non-stereotyped cohorts by matching VH CDR3 length led to similar conclusions. Within stereotyped cases there was a wide distribution of VHR, ranging from 55.6% to 0.1%. Restricting VH CDR3 lengths to "short" (5 - ≤13), "medium" (13.1 - ≤20) and "long" (20.1 - ≤28), the corresponding VHR increased monotonically with length (1.1, 8.2, and 11.9% respectively). Notably, subsets showing elevated VH replacement included better prognosis subsets, #4, 77 and 201 (23.8, 22.1, and 28.6%, respectively). Among low VHR frequency subsets were those associated with worse prognosis, #1, 2, 5, 6, 8, 9 and 10 (VHR frequencies: 0.2, 0.1, 0.9, 2.3, 7.7, 9.0 %, respectively). This was most strikingly exhibited by subsets #1 and #2, both of which comprise patients with poor clinical courses. Each of these sets of sequences displayed virtually no examples of VHR (0.2 and 0.1%, respectively). This might be predicted because these two subsets have relatively short VH CDR3 lengths (subset #1: 13 aa; subset #2: 9 aa), based on the length association mentioned above. Detailed analyses of the presence of footprints and the position of these in the rearranged IGHV-D-J indicated that for some subsets, certain signature VH CDR3 amino acids could be the result of VHR. For example in subset #201, sequence analysis suggests that VHR is responsible for an arginine and for a glutamine in the 5' portion of the VH CDR3. Similarly, VHR may craft the characteristic glutamine on the 5' end of the subset #6 VH CDR3. Thus, our studies indicate that, as a whole, CLL IGHV-D-J sequences use VHR at a frequency comparable to that of normal B cells and significantly less than that of non-stereotyped rearrangements. However, certain stereotyped cases are dramatically enriched for evidence of VHR. Moreover among these cases, the footprints found in the VH CDR3s of stereotyped cases can be shown to directly code for signature amino acids in VH CDR3s. Finally, stereotyped cases with high levels of VHR tend to be those with better clinical courses, whereas those worse outcome stereotyped cases exhibit less evidence for this process. This latter finding is consistent with the concept that VHR is one of the molecular mechanisms used by developing B cells to edit BCRs having high affinity for autoantigens. Since many CLL BCRs are autoreactive, including those found to have high levels of VHR such as subset #4, this implies a fundamental defect in tolerance mechanisms in those normal B cells that eventually became leukemic. Disclosures Agathangelidis: Gilead: Research Funding. Hadzidimitriou:Janssen: Honoraria, Research Funding; Gilead: Research Funding; Abbvie: Research Funding. Ghia:AbbVie, Inc: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Sunesis: Honoraria, Research Funding. Stamatopoulos:Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Chiorazzi:AR Pharma: Equity Ownership; Janssen, Inc: Consultancy.

Description: Using next-generation sequencing (NGS), we recently documented the clonal architecture of the T cell repertoire in treatment-naive chronic lymphocytic leukemia (CLL), with ample immunogenetic evidence indicating selection by restricted antigens. Our preliminary NGS study in 16 patients pre- and 3-month post-treatment indicated a differential impact of standard chemoimmunotherapy (FCR) versus B cell receptor signaling inhibitors (BcRi) on CLL T cells. Prompted by these observations, here we sought to comprehensively assess CLL T cell repertoire changes over treatment in relation to both treatment type and clinical response by combining NGS immunoprofiling, flow cytometry and functional assays. NGS profiling of the T cell receptor (TR) gene repertoire was performed in 28 CLL patients who received FCR (n=9), ibrutinib (IB, n=15) and/or rituximab-idelalisib (R-ID, n=10) at successive timepoints (pre, +3mo, +9mo and at deepest clinical response, total samples: n=113). TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end NGS. Raw reads were processed through a purpose-built, validated bioinformatics pipeline, culminating to 20,347,768 productive, filtered-in TRB sequences (median 155,479/sample). For repertoire analysis, clonotypes (i.e. rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 sequence) were considered (median 11,420 distinct clonotypes/sample). All cases displayed significant clonal T cell expansions both pre- and post-treatment [median clonality, measured as the cumulative frequency of the 10 most expanded (major) clonotypes/sample: 30.3% and 39.6%, respectively]. Median clonality significantly increased at +3mo in the FCR (29.0% to 46.9%, p

Description: Somatic hypermutation (SHM) features in a series of 1967 immunoglobulin heavy chain gene (IGH) rearrangements obtained from patients with chronic lymphocytic leukemia (CLL) were examined and compared with IGH sequences from non-CLL B cells available in public databases. SHM analysis was performed for all 1290 CLL sequences in this cohort with less than 100% identity to germ line. At the cohort level, SHM patterns were typical of a canonical SHM process. However, important differences emerged from the analysis of certain subgroups of CLL sequences defined by: (1) IGHV gene usage, (2) presence of stereotyped heavy chain complementarity-determining region 3 (HCDR3) sequences, and (3) mutational load. Recurrent, “stereotyped” amino acid changes occurred across the entire IGHV region in CLL subsets carrying stereotyped HCDR3 sequences, especially those expressing the IGHV3-21 and IGHV4-34 genes. These mutations are underrepresented among non-CLL sequences and thus can be considered as CLL-biased. Furthermore, it was shown that even a low level of mutations may be functionally relevant, given that stereotyped amino acid changes can be found in subsets of minimally mutated cases. The precise targeting and distinctive features of somatic hypermutation (SHM) in selected subgroups of CLL patients provide further evidence for selection by specific antigenic element(s).

Description: The chronic lymphocytic leukemia (CLL) immunoglobulin repertoire is biased and characterized by the existence of subsets of cases with closely homologous (“stereotyped”) complementarity-determining region 3 (CDR3) sequences. In the present series, 201 (21.9%) of 916 patients with CLL expressed IGHV genes that belonged to 1 of 48 different subsets of sequences with stereotyped heavy chain (H) CDR3. Twenty-six subsets comprised 3 or more sequences and were considered “confirmed.” The remaining subsets comprised pairs of sequences and were considered “potential”; public database CLL sequences were found to be members of 9 of 22 “potential” subsets, thereby allowing us to consider them also “confirmed.” The chance of belonging to a subset exceeded 35% for unmutated or selected IGHV genes (eg, IGHV1-69/3-21/4-39). Comparison to non-CLL public database sequences showed that HCDR3 restriction is “CLL-related.” CLL cases with selected stereotyped immunoglobulins (IGs) were also found to share unique biologic and clinical features. In particular, cases expressing stereotyped IGHV4-39/IGKV1-39-1D-39 and IGHV4-34/IGKV2-30 were always IgG-switched. In addition, IGHV4-34/IGKV2-30 patients were younger and followed a strikingly indolent disease, contrasting other patients (eg, those expressing IGHV3-21/IGLV3-21) who experienced an aggressive disease, regardless of IGHV mutations. These findings suggest that a particular antigen-binding site can be critical in determining the clinical features and outcome for at least some CLL patients.

Description: The immunoglobulin heavy chain variable (IGHV) gene repertoire in CLL is biased and uniquely characterized by the existence of subsets of cases with “stereotyped” heavy chain complementarity-determining region 3 (HCDR3) sequences. As previously shown, HCDR3 stereotypy may have important pathogenetic and clinical implications, at least for certain subsets of CLL patients. However, the detection of stereotypy has so far been hindered, mainly due to the lack of suitable computational tools. We developed new methods for identification of sequence patterns within HCDR3 amino acid (AA) sequences using a sophisticated combinatorial pattern discovery algorithm. Included in the analysis were HCDR3 sequences from 2,845 CLL patients from our collaborating institutions, as well as 5,344 non-CLL sequences from public databases, for a total of 8,189. The identified patterns were subjected to a multiple and strict filtering process, based on the following criteria: sequence relatedness; location (offset) within HCDR3; and HCDR3 AA length. Clustering of sequences based on the filtered HCDR3 patterns revealed that the CLL IG repertoire can be distinguished in two broad categories: the first includes cases with heterogeneous B cell receptors (BCRs) (non-clustered cases) while the second is characterized by remarkable BCR stereotypy (clustered cases). In particular, 783/2,845 CLL sequences (27.5%) were placed in 339 ground-level clusters. Common sequences among these ground-level clusters allowed their progressive grouping in clusters at higher levels of hierarchy. High-level clusters were characterized by striking IGHV repertoire restriction, with only six IGHV genes (1–69, 1–3, 1–2, 3–21, 4–34, 4–39) accounting for 〉80% (382/459) of cases. While ground-level clusters provided a high-resolution picture of HCDR3 stereotypy, high-level clusters were considerably larger in size (up to 86 sequences each) and able to capture and describe more distant sequence relationships in the form of more widely shared sequence patterns. In particular, they were defined by patterns characterized by just a few critically positioned residues, reminiscent of the receptors expressed by cells participating in innate immune responses: due to this remarkable structural conservation, we consider them to be BCR “archetypes”. To test the hypothesis that sequence relatedness between IGHV genes may have structural and functional meaning for the IGHV repertoire in CLL, we also constructed sequence distance trees of functional human IGHV genes. Examination of the tree for the IGHV3 gene subgroup revealed a branching that was reflected in the repertoires of clustered vs. non-clustered CLL sequences. In particular, IGHV3-21, the foremost example of a gene with a propensity to be used in clustered rearrangements in CLL, belongs to a branch clearly distinct from other branches that include, for instance, the IGHV3-23, IGHV3-30, IGHV3-33 and IGHV3-7 genes, which were essentially absent from the repertoire of CLL sequences in high-level clusters; a similar case was also evident among IGHV1 subgroup genes. On these grounds, we argue that CLL cases with clustered (i.e. stereotyped) vs. non-clustered (i.e. heterogeneous) BCRs could derive from different progenitor cell populations evolutionarily adapted to particular antigenic challenges. In particular, prompted by the fact that clustered cases were found to express a limited set of highly conserved BCRs, we propose that in such cases the clonogenic progenitors may originate from a B cell population intermediate between a true innate immune system and the conventional adaptive B cell immune system, similar to what has been previously suggested for mouse B1 cells.