Host virus and pneumococcus-specific immune responses in high-count monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia: implications for disease progression.

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 B-cell 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.

Subjects with chronic lymphocytic leukaemia-like B-cell clones with stereotyped B-cell receptors frequently show MDS-associated phenotypes on myeloid cells.

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.

Molecular and cytogenetic characterization of expanded B-cell clones from multiclonal versus monoclonal B-cell chronic lymphoproliferative disorders.

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.

Monoclonal B-cell lymphocytosis (MBL) with normal lymphocyte counts is associated with decreased numbers of normal circulating B-cell subsets.

Monoclonal B-cell lymphocytosis (MBL) with normal lymphocyte counts is associated with decreased numbers of normal circulating B-cell subsets.Little is known about the distribution of normal lymphoid cells and their subsets in the peripheral blood (PB) of subjects with monoclonal B-cell lymphocytosis (MBL). In our study, we compared the absolute number of PB lymphoid cells and their subpopulations in 95 MBL cases with normal lymphocyte counts vs. 617 age-/sex-matched non-MBL healthy subjects (controls), using highly sensitive flow cytometry. MBL cases showed significantly reduced numbers of normal circulating B-cells, at the expense of immature and naive B-cells; in addition, CD4+CD8+ double-positive T-cells and CD8+ T-cells were significantly lower and higher vs. controls, respectively. Moreover, most normal B-cell subsets were significantly decreased in PB at >1% MBL-counts, vs. "low-count" MBL cases, and lower amounts of immature/naive B-cells were detected in biclonal (particularly in cases with coexisting CLL-like- and non-CLL-like B-cell clones) vs. monoclonal MBL subjects. In summary, our results show imbalanced (reduced) absolute numbers of recently produced normal circulating B-cells (e.g., immature and naive B-cells) in MBL, which becomes more pronounced as the MBL cell count increases.

Hematological Findings in Lysosomal Storage Disorders: A Perspective from the Medical Laboratory.

Lysosomal storage disorders (LSDs) are a group of rare and genetic diseases produced by mutations in genes coding for proteins involved in lysosome functioning. Protein defect leads to the lysosomal accumulation of undegraded macromolecules including glycoproteins, glycosaminoglycans, lipids, and glycogen. Depending on the stored substrate, several pathogenic cascades may be activated leading to multisystemic and progressive disorders affecting the brain, eye, ear, lungs, heart, liver, spleen, kidney, skin, or bone. In addition, for some of these disorders, hematological findings have been also reported. In this paper, we review the major hematological alterations in LSDs based on 56 case reports published between 2010 and 2020. Hematological alterations were reported in sphingolipidosis, mucopolysaccharidoses, mucolipidoses, neuronal ceroid lipofuscinosis, glycogenosis, glycoproteinosis, cystinosis, and cholesteryl ester storage disease. They were reported alterations in red cell linage and leukocytes, such as anemia and morphology changes in eosinophils, neutrophils, monocytes, and lymphocytes. In addition, changes in platelet counts (thrombocytopenia) and leukocyte abnormalities on non-peripheral blood samples were also reported for some LSDs. Although in most of the cases these hematological alterations are not pathognomonic of a specific disease or group of LSDs, since they can be easily identified in general clinical laboratories, their identification may contribute to the diagnosis of these disorders. In this sense, we hope that this review contributes to the awareness of the importance of hematological alterations in the diagnosis of LSDs.

Age- and Sex-Matched Normal Leukocyte Subset Ranges in the General Population Defined with the EuroFlow Lymphocyte Screening Tube (LST) for Monoclonal B-Cell Lymphocytosis (MBL) vs. Non-MBL Subjects.

Reference ranges of blood-circulating leukocyte populations by, e.g., age and sex, are required for monitoring immune-cell kinetics. Most previous reports in which flow cytometry has been used to define the reference ranges for leukocyte counts included a limited number of donors and/or cell populations and/or did not consider age and sex simultaneously. Moreover, other factors not previously considered in the definition of normal ranges, such as the presence of chronic-lymphocytic-leukemia (CLL)-like low-count monoclonal B-cell lymphocytosis (MBLlo), might also be associated with an altered distribution of leukocytes in blood in association with an immunodeficiency and increased risk of infection and cancer. Here, we established reference cell-count ranges for the major populations of leukocytes in blood of non-MBL and MBLlo adult Caucasians matched by age and sex using the EuroFlow Lymphocyte Screening Tube (LST). A total of 706 Caucasian adult donors­622 non-MBL and 84 MBLlo­were recruited from the general population. Among non-MBL donors, the total leukocyte, neutrophil, basophil dendritic cell and monocyte counts remained stable through adulthood, while the absolute numbers of T- and B-cell populations and plasma cells decreased with age. The number of eosinophils and NK-cell increased over time, with clear differences according to sex for certain age ranges. In MBLlo subjects, few differences in the absolute cell counts by age (vs. non-MBL) were observed, and MBLlo men and women showed similar trends to non-MBL subjects except for the B-cell count drop observed in >70 y-men, which was more pronounced in MBLlo vs. non-MBL controls. Building robust age- and sex-matched reference ranges for the most relevant immune-cell populations in the blood of non-MBL donors is essential to appropriately identify an altered immune status in different clinical settings and highlight the altered immune-cell profiles of MBLlo subjects.

Increased frequency (12%) of circulating chronic lymphocytic leukemia-like B-cell clones in healthy subjects using a highly sensitive multicolor flow cytometry approach.

Monoclonal B-cell lymphocytosis (MBL) indicates the presence of less than 5 x 10(9)/L circulating monoclonal B cells in otherwise healthy subjects. Recently, it has been reported that circulating chronic lymphocytic leukemia (CLL)-like B cells can be detected using 4- or 5-multicolor flow cytometry in 5% to 7% of adults with normal lymphocyte counts. We investigated the frequency of circulating monoclonal B cells in 608 healthy subjects older than 40 years with normal blood counts, using a highly sensitive 8-color flow cytometry approach and systematic screening for total PB leukocyte count higher than 5 x 10(6). We show that the frequency of PB monoclonal B cells is markedly higher than previously reported (12% for CLL-like B cells, found at frequencies of 0.17 +/- 0.13 x 10(9) cells/L), the incidence progressively increasing with age. Most cases (62%) showed clonal B-cell levels below the maximum sensitivity of the techniques described by others (< 0.01%), supporting the notion that detection of MBL may largely depend on the sensitivity of the flow cytometry approach used.

A systematic approach for peptide characterization of B-cell receptor in chronic lymphocytic leukemia cells.

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.

Combined patterns of IGHV repertoire and cytogenetic/molecular alterations in monoclonal B lymphocytosis versus chronic lymphocytic leukemia.

BACKGROUND: Chronic lymphocytic leukemia (CLL)-like monoclonal B lymphocytosis (MBL) with (MBL(hi)) or without (MBL(lo)) 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 MBL(lo) 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 MBL(hi) 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 MBL(lo) 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.

Common infectious agents and monoclonal B-cell lymphocytosis: a cross-sectional epidemiological study among healthy adults.

BACKGROUND: Risk factors associated with monoclonal B-cell lymphocytosis (MBL), a potential precursor of chronic lymphocytic leukaemia (CLL), remain unknown. METHODS: Using a cross-sectional study design, we investigated demographic, medical and behavioural risk factors associated with MBL. "Low-count" MBL (cases) were defined as individuals with very low median absolute count of clonal B-cells, identified from screening of healthy individuals and the remainder classified as controls. 452 individuals completed a questionnaire with their general practitioner, both blind to the MBL status of the subject. Odds ratios (OR) and 95% confidence interval (CI) for MBL were estimated by means of unconditional logistic regression adjusted for confounding factors. RESULTS: MBL were detected in 72/452 subjects (16%). Increasing age was strongly associated with MBL (P-trend<0.001). MBL was significantly less common among individuals vaccinated against pneumococcal or influenza (OR 0.49, 95% confidence interval (CI): 0.25 to 0.95; P-value=0.03 and OR: 0.52, 95% CI: 0.29 to 0.93, P-value=0.03, respectively). Albeit based on small numbers, cases were more likely to report infectious diseases among their children, respiratory disease among their siblings and personal history of pneumonia and meningitis. No other distinguishing epidemiological features were identified except for family history of cancer and an inverse relationship with diabetes treatment. All associations described above were retained after restricting the analysis to CLL-like MBL. CONCLUSION: Overall, these findings suggest that exposure to infectious agents leading to serious clinical manifestations in the patient or its surroundings may trigger immune events leading to MBL. This exploratory study provides initial insights and directions for future research related to MBL, a potential precursor of chronic lymphocytic leukaemia. Further work is warranted to confirm these findings.

Non-CLL-like monoclonal B-cell lymphocytosis in the general population: prevalence and phenotypic/genetic characteristics.

BACKGROUND: Monoclonal B-cell lymphocytosis (MBL) indicates <5 × 10(9) peripheral blood (PB) clonal B-cells/L in healthy individuals. In most cases, MBL cells show similar phenotypic/genetic features to chronic lymphocytic leukemia cells-CLL-like MBL-but little is known about non-CLL-like MBL. METHODS: PB samples from 639 healthy individuals (46% men/54% women) >40 years old (62 ± 13 years) with normal lymphocyte counts (2.1 ± 0.7 × 10(9)/L) were immunophenotyped using high-sensitive flow cytometry, based on 8-color stainings and the screening for >5 × 10(6) total PB leukocytes. RESULTS: Thirteen subjects (2.0%; 9 males/4 females, aged 73 ± 10 years; absolute lymphocyte count: 2.4 ± 0.8 × 10(9)/L) showed a non-CLL-like clonal B-cell population, whose frequency clearly increased with age: 0.4%, 3%, and 5.4% of subjects aged 40-59, 60-79, and ≥80 years, respectively. One single B-cell clone was detected in 9/13 cases, while two B-cell clones were found in 4/13 (n = 17 MBL populations). Nine MBL cell populations showed a CD5(-) phenotype (usually overlapping with marginal zone-derived (MZL) or lymphoplasmacytic (LPL) non-Hodgkin lymphoma (NHL) B-cells, or an unclassifiable NHL), but CD5(-/+d) (n = 3) and CD5(+) (n = 3 non-CLL-like MBL, consistent with a mantle-cell lymphoma (MCL)-like phenotype, and n = 2 CLL-like) MBL were also identified; iFISH supported the diagnosis in most cases. No preferential IGHV usage of B-cell receptor could be found. Twelve cases reevaluated at month +12 showed circulating clonal B-cells, at mean levels significantly higher than those initially detected. CONCLUSIONS: Non-CLL-like MBL cases frequently show biclonality, in association with MZL-, LPL-, MCL-like, or unclassifiable phenotypic profiles. As with CLL-like MBL, the frequency of non-CLL-like MBL increases with age, with a clear predominance of males.

Commentary: Comparison of current flow cytometry methods for monoclonal B cell lymphocytosis detection.

Monoclonal B cell lymphocytosis (MBL) is now recognized as the B-lymphocyte analogue of a monoclonal gammopathy of unknown significance. MBL can be the precursor of chronic lymphocytic leukemia or associated with non-Hodgkin's lymphoma. It may be associated with an autoimmune abnormality or be related to aging (immunosenescence). The combination of available new fluorochrome-conjugated monoclonal antibody reagents, multilaser instrumentation, and improved software tools have led to a new level of multicolor analysis of MBL. Presently, several centers, including the University of Salamanca (Spain), Duke University (Durham, NC), Mayo Clinic (Rochester, MN), and the National Cancer Institute (Bethesda, MD) in conjunction with the Genetics and Epidemiology of Familial chronic lymphocytic leukemia Consortium, the Food and Drug Administration (Bethesda, MD), and the Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry (Atlanta, GA) in collaboration with Saint Luke's Hospital (Kansas City, MO), the Università Vita-Salute San Raffaele in Milan (Italy), and Leeds Teaching Hospital (UK) are all actively conducting studies on MBL. This commentary is an updated summary of the current methods used in these centers. It is important to note the diversity of use in reagents, instruments, and methods of analysis. Despite this diversity, there is a consensus in what constitutes the diagnosis of MBL and its subtypes. There is also an emerging consensus on what the next investigative steps should be.