Search

Molecular techniques are the gold standard method for the diagnosis of AML with mutated nucleophosmin gene (NPM1mut). However, their worldwide availability is limited and they provide limited insight into disease heterogeneity. Hence, surrogate markers of NPM1mut are used for fast diagnostic screening of the disease [1], including, among others, immunohistochemical detection of cytoplasmic NPM1 (NPM1c) [2], cup-like nuclear morphology [3], normal karyotype, and/or recurrent flow cytometry profiles -e.g., CD34 negativity, and/or a phenotype resembling acute promyelocytic leukemia (APL)- [4]. Nevertheless, some of these methods are also not widely available, they show limited sensitivity (e.g., low or absent NPM1c expression, particularly among monoblastic/monocytic AML-NPM1mut) [5], frequently lack standardized procedures [1], and they might also bring limited information about disease heterogeneity.

The HCDR3 sequences of the B-cell receptor (BCR) undergo constraints in length, amino acid use, and charge during maturation of B-cell precursors and after antigen encounter, leading to BCR and antibodies with high affinity to specific antigens. Chronic lymphocytic leukemia consists of an expansion of B-cells with a mixed immature and “antigen-experienced” phenotype, with either a mutated (M-CLL) or unmutated (U-CLL) tumor BCR, associated with distinct patient outcomes. Here, we investigated the hydropathy index of the BCR of 138 CLL patients and its association with the IGHV mutational status and patient outcome. Overall, two clearly distinct subgroups of M-CLL patients emerged, based on a neutral (mean hydropathy index of -0.1) vs. negatively charged BCR (mean hydropathy index of -1.1) with molecular features closer to those of B-cell precursors and peripheral/mature B-cells, respectively. Despite that M-CLL with neutral HCDR3 did not show traits associated with a mature B-cell repertoire, important differences in IGHV gene usage of tumor cells and patient outcome were observed in this subgroup of patients once compared to both U-CLL and M-CLL with negatively charged HCDR3 sequences. Compared to M-CLL with negatively charged HCDR3 sequences, M-CLL with neutral HCDR3 sequences showed predominance of men, more advanced stages of the disease, and a greater frequency of genetic alterations—e.g., del(17p)—together with a higher rate of disease progression and shorter time to therapy (TTT), independently of other prognostic factors. Our data suggest that the hydropathy index of the HCDR3 sequences of CLL cells allows the identification of a subgroup of M-CLL with intermediate prognostic features between U-CLL and the more favorable subgroup of M-CLL with a negatively charged BCR.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries, which is characterized by the accumulation of mature CD5+/CD20lo/CD23+ clonal B-cells in peripheral blood (PB), bone marrow (BM), and other lymphoid tissues [1]. Currently, it is well-established that CLL is systematically preceded by a pre-leukemic stage, known as monoclonal B-cell lymphocytosis (MBL) [2]; MBL includes both low-count (MBLlo) and high-count MBL (MBLhi), depending on the number of PB clonal B-cells (<0.5 × 109/L and ≥0.5 × 109/L, respectively) detected [3], the former being a highly prevalent condition in adults (≈25% of individuals >70 y) [4, 5]. The biological and clinical significance of CLL-like clonal B-cells in PB of otherwise healthy individuals (MBLlo) has not been fully elucidated [6,7,8]. Recently, we have reported a very low rate of transformation of MBLlo to MBLhi/CLL, after 7 years of follow-up [8]. In contrast, we found a higher frequency of deaths in MBLlo subjects vs. age- and sex-matched non-MBL healthy adults from the same geographical area; among the former subjects, infection was an overrepresented cause of death (21% vs. 2%, respectively) [8]. This is in line with previous studies showing an ≈3-fold increased risk of infection in both MBLhi and CLL patients, in whom infections also represent a major cause of death [9, 10].

Low-count monoclonal B-cell lymphocytosis is defined by the presence of very low numbers of circulating clonal B cells, usually phenotypically similar to chronic lymphocytic leukemia cells, whose biological and clinical significance remains elusive. Herein, we re-evaluated 65/91 low-count monoclonal B-cell lymphocytosis cases (54 chronic lymphocytic leukemia-like and 11 non-chronic lymphocytic leukemia-like) followed-up for a median of seven years, using high-sensitivity flow cytometry and interphase fluorescence in situ hybridization. Overall, the clone size significantly increased in 69% of low-count monoclonal B-cell lymphocytosis cases, but only one subject progressed to high-count monoclonal B-cell lymphocytosis. In parallel, the frequency of cytogenetic alterations increased over time (32% vs. 61% of cases, respectively). The absolute number of the major T-cell and natural killer cell populations also increased, but only among chronic lymphocytic leukemia-like cases with increased clone size vs. age- and sex-matched controls. Although progression to chronic lymphocytic leukemia was not observed, the overall survival of low-count monoclonal B-cell lymphocytosis individuals was significantly reduced vs. non-monoclonal B-cell lymphocytosis controls (P=0.03) plus the general population from the same region (P≤0.001), particularly among females (P=0.01); infection and cancer were the main causes of death in low-count monoclonal B-cell lymphocytosis. In summary, despite the fact that mid-term progression from low-count monoclonal B-cell lymphocytosis to high-count monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia appears to be unlikely, these clones persist at increased numbers, usually carrying more genetic alterations, and might thus be a marker of an impaired immune system indirectly associated with a poorer outcome, particularly among females.

[Background]: Humoral immunocompetence develops stepwise throughout life and contributes to individual susceptibility to infection, immunodeficiency, autoimmunity, and neoplasia. Immunoglobulin heavy chain (IgH) isotype serum levels can partly explain such age-related differences, but their relationship with the IgH isotype distribution within memory B-cell (MBC) and plasma cell (PCs) compartments remains to be investigated. [Objective]: We studied the age-related distribution of MBCs and PCs expressing different IgH isotypes in addition to the immature/transitional and naive B-cell compartments. [Methods]: B-cell and PC subsets and plasma IgH isotype levels were studied in cord blood (n = 19) and peripheral blood (n = 215) from healthy donors aged 0 to 90 years by using flow cytometry and nephelometry, respectively. [Results]: IgH-switched MBCs expressing IgG, IgG, IgG, IgA, and IgA were already detected in cord blood and newborns at very low counts, whereas CD27IgMIgD MBCs only became detectable at 1 to 5 months and remained stable until 2 to 4 years, and IgD MBCs peaked at 2 to 4 years, with both populations decreasing thereafter. MBCs expressing IgH isotypes of the second immunoglobulin heavy chain constant region (IGHC) gene block (IgG, IgG, and IgA) peaked later during childhood (2-4 years), whereas MBCs expressing third IGHC gene block immunoglobulin isotypes (IgG, IgG, and IgA) reached maximum values during adulthood. PCs were already detected in newborns, increasing in number until 6 to 11 months for IgM, IgG, IgG, IgG, IgA, and IgA; until 2 to 4 years for IgD; and until 5 to 9 years for IgG and decreasing thereafter. For most IgH isotypes (except IgD and IgG), maximum plasma levels were reached after PC and MBC counts peaked. [Conclusions]: PC counts reach maximum values early in life, followed by MBC counts and plasma IgH isotypes. Importantly, IgH isotypes from different IGHC gene blocks show different patterns, probably reflecting consecutive cycles of

A wide variety of immunoglobulins (Ig) is produced by the immune system thanks to different mechanisms (V(D)J recombination, somatic hypermutation, and antigen selection). The profiling of Ig sequences (at both DNA and peptide levels) are of great relevance to developing targeted vaccines or treatments for specific diseases or infections. Thus, genomics and proteomics techniques (such as Next- Generation Sequencing (NGS) and mass spectrometry (MS)) have notably increased the knowledge in Ig sequencing and serum Ig peptide profiling in a high-throughput manner. However, the peptide characterization of membrane-bound Ig (e.g., B-cell receptors, BCR) is still a challenge mainly due to the poor recovery of mentioned Ig. Herein, we have evaluated three different sample processing methods for peptide sequencing of BCR belonging to chronic lymphocytic leukemia (CLL) B cells identifying up to 426 different peptide sequences (MS/MS data are available via ProteomeXchange with identifier PXD004466). Moreover, as a consequence of the results here obtained, recommended guidelines have been described for BCR-sequencing of B-CLL samples by MS approaches. For this purpose, an in-house algorithm has been designed and developed to compare the MS/MS results with those obtained by molecular biology in order to integrate both proteomics and genomics results and establish the steps to follow when sequencing membrane-bound Ig by MS/MS.

Patients diagnosed with chronic lymphocytic leukemia (CLL) display a high incidence of infections due to an associated immunodeficiency that includes hypogammaglobulinemia. A higher risk of infections has also been recently reported for high-count monoclonal B-cell lymphocytosis, while no information is available in low-count monoclonal B-cell lymphocytosis. Here, we evaluated the status of the humoral immune system in patients with chronic lymphocytic leukemia (n=58), as well as in low- (n=71) and high- (n=29) count monoclonal B-cell lymphocytosis versus healthy donors (n=91). Total free plasma immunoglobulin titers and specific levels of antibodies against cytomegalovirus, Epstein-Barr virus, influenza and S.pneumoniae were measured by nephelometry and ELISA-based techniques, respectively. Overall, our results show that both CLL and high-count monoclonal Bcell lymphocytosis patients, but not low-count monoclonal B-cell lymphocytosis subjects, present with relatively high levels of antibodies specific for the latent viruses investigated, associated with progressively lower levels of S.pneumoniae-specific immunoglobulins. These findings probably reflect asymptomatic chronic reactivation of humoral immune responses against host viruses associated with expanded virus-specific antibody levels and progressively decreased protection against other micro-organisms, denoting a severe humoral immunodeficiency state not reflected by the overall plasma immunoglobulin levels. Alternatively, these results could reflect a potential role of ubiquitous viruses in the pathogenesis of the disease. Further analyses are necessary to establish the relevance of such asymptomatic humoral immune responses against host viruses in the expansion of the tumor B-cell clone and progression from monoclonal B-cell lymphocytosis to CLL.

An increasing body of evidence suggests the potential occurrence of antigen encounter by the cell of origin in chronic lymphocytic leukaemia (CLL) and CLL-like monoclonal B-cell lymphocytosis (MBL). However, the scenario in which this event might occur remains unknown. In order to gain insight into this scenario we investigated the molecular, cytogenetic and haematological features of 223 CLL-like (n = 84) and CLL (n = 139) clones with stereotyped (n = 32) versus non-stereotyped (n = 191) immunoglobulin heavy chain variable region (IGHV) amino acid sequences. Overall, stereotyped CLL-like MBL and CLL clones showed a unique IGHV profile, associated with higher IGHV1 and lower IGHV3 gene family usage (P = 0·03), longer IGHV complementary determining region 3 (HCDR3) sequences (P = 0·007) and unmutated IGHV (P < 0·001) versus non-stereotyped clones. Whilst the overall size of the stereotyped B-cell clones in peripheral blood did not appear to be associated with the CLL-related cytogenetic profile of B-cells (P > 0·05), it did show a significant association with the presence of myelodysplastic syndrome (MDS)-associated immunophenotypes on peripheral blood neutrophils and/or monocytes (P = 0·01). Altogether our results point to the potential involvement of different selection forces in the expansion of stereotyped vs. non-stereotyped CLL and CLL-like MBL clones, the former being potentially favoured by an underlying altered haematopoiesis.

Chronic antigen-stimulation has been recurrently involved in the earlier stages of monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The expansion of two or more B-cell clones has frequently been reported in individuals with these conditions; potentially, such coexisting clones have a greater probability of interaction with common immunological determinants. Here, we analyzed the B-cell receptor repertoire and molecular profile, as well as the phenotypic, cytogenetic and hematologic features, of 228 chronic lymphocytic leukemia-like and non-chronic lymphocytic leukemia-like clones comparing multiclonal (n=85 clones from 41 cases) versus monoclonal (n=143 clones) monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The B-cell receptor of B-cell clones from multiclonal cases showed a slightly higher degree of HCDR3 homology than B-cell clones from mono clonal cases, in association with unique hematologic (e.g. lower B-lymphocyte counts) and cytogenetic (e.g. lower frequency of cytogenetically altered clones) features usually related to earlier stages of the disease. Moreover, a subgroup of coexisting B-cell clones from individual multiclonal cases which were found to be phylogenetically related showed unique molecular and cytogenetic features: they more frequently shared IGHV3 gene usage, shorter HCDR3 sequences with a greater proportion of IGHV mutations and del(13q14.3), than other unrelated B-cell clones. These results would support the antigen-driven nature of such multiclonal B-cell expansions, with potential involvement of multiple antigens/epitopes.

[Background]:Chronic lymphocytic leukemia (CLL)-like monoclonal B lymphocytosis (MBL) with (MBLhi) or without (MBLlo) absolute B-lymphocytosis precedes most CLL cases,the specific determinants for malignant progression remaining unknown. [Methodology/Principal Findings]:For this purpose, simultaneous iFISH and molecular analysis of well-established cytogenetic alterations of chromosomes 11, 12, 13, 14 and 17 together with the pattern of rearrangement of the IGHV genes were performed in CLL-like cells from MBL and CLL cases. Our results based on 78 CLL-like MBL and 117 CLL clones from 166 subjects living in the same geographical area, show the existence of three major groups of clones with distinct but partially overlapping patterns of IGHV gene usage, IGHV mutational status and cytogenetic alterations. These included a group enriched in MBLlo clones expressing specific IGHV subgroups (e.g. VH3-23) with no or isolated good-prognosis cytogenetic alterations, a second group which mainly consisted of clinical MBLhi and advanced stage CLL with a skewed but different CLL-associated IGHV gene repertoire (e.g. VH1-69), frequently associated with complex karyotypes and poor-prognosis cytogenetic alterations, and a third group of clones with intermediate features, with prevalence of mutated IGHV genes, and higher numbers of del(13q)+ clonal B-cells. [Conclusions/Significance]: These findings suggest that the specific IGHV repertoire and IGHV mutational status of CLL-like B-cell clones may modulate the type of cytogenetic alterations acquired, their rate of acquisition and/or potentially also their clinical consequences. Further long-term follow-up studies investigating the IGHV gene repertoire of MBLlo clones in distinct geographic areas and microenvironments are required to confirm our findings and shed light on the potential role of some antigen-binding BCR specificities contributing to clonal evolution.