Novel Analysis of Clonal Diversification in Blood B Cell and Bone Marrow Plasma Cell Clones in Immunoglobulin Light Chain Amyloidosis

Immunoglobulin light chain amyloidosis (AL) is characterized by a limited clonal expansion of plasma cells and amyloid formation. Here, we report restriction in the diversity of VL gene usage with a dominance of clonally related B cells in the peripheral blood (PB) isotype-specific repertoire of AL patients. A rigorous quantification of lineage trees reveals presence of intraclonal variations in the PB clones compared to the bone marrow (BM) clones, which suggests a common precursor that is still subject to somatic mutation. When compared to normal BM and PB B cells, AL clones showed significant but incomplete impairment of antigenic selection, which could not be detected by conventional R and S mutation analysis. Therefore, graphical analysis of B cell lineage trees and mathematical quantification of tree properties provide novel insights into the process of B cell clonal evolution in AL.     

Quantitative analysis of clonal bone marrow CD19+ B cells: use of B cell lineage trees to delineate their role in the pathogenesis of light chain amyloidosis

Light chain amyloidosis (AL) is a bone marrow (BM) plasma cell neoplasia with systemic deposition of Ig light chain amyloid fibrils. Here, we report the identification of clonal CD19 B cells in the BM and the use of a novel mathematical algorithm to generate B cell lineage trees of the clonal CD19 B cells and CD138 plasma cells from the BM of AL patients to delineate the relationship between these two clonal populations. The CD19+ clonal B cells in the BM of AL patients related to the clonal plasma cells represent a pre-plasma cell precursor population. The B cell lineage trees from AL patients also show significant differences in clonal diversification and antigenic selection compared to clones from normal, healthy controls. These data provide a robust example of the use of graphical quantification methods in delineating the role of neoplastic precursors in the pathogenesis of hematopoietic malignancies.     

Bone marrow findings correlate with clinical outcome in systemic AL amyloidosis patients

AIMS: Bone marrow sampling is a key investigation in the work-up of amyloid light chain (AL) amyloidosis, but the relationship between bone marrow findings and the varied phenotype and clinical outcome of AL amyloidosis is unclear. The aim was to determine if bone marrow pathological parameters at diagnosis were related to clinical behaviour in AL amyloidosis patients. METHODS AND RESULTS: Bone marrow findings, clinical features and outcome of 80 patients referred with a diagnosis of systemic AL amyloidosis were evaluated; six patients were subsequently excluded due to re-categorization as other forms of amyloidosis. At latest follow-up (median 66 months), 11 of the 18 patients with no identifiable bone marrow neoplastic cells (61%) versus only seven of the 56 patients with neoplastic plasma cells or non-Hodgkin's lymphoma (13%) were alive (P = 0.0046). However, neither the quantity of the neoplastic cells nor the serum light chain levels were correlated with amyloid burden or patient survival. CONCLUSIONS: Identification of a neoplastic population in the bone marrow of AL amyloidosis patients by histology and immunohistochemistry correlates with poor outcome; however, the neoplastic cell burden is not prognostically significant, suggesting that additional factors are important in determining disease behaviour in AL amyloidosis.     

Normal and clonal B lineage cells can be distinguished by their differential expression of B cell antigens and adhesion molecules in peripheral blood from multiple myeloma (MM) patients—diagnostic and clinical implications

Human MM is a haematologic disorder characterized by the accumulation of malignant plasma cells (PC), primarily in the bone marrow (BM). Although these cells characteristically home to the BM, in recent years several groups have detected the presence of related malignant B cells in the peripheral blood (PB) which could be implicated in the progression and spread of the disease. However, the proportion and origin of these clonotypic circulating B cells is still controversial. In this study, using a triple-staining flow cytometric procedure and a whole blood lysis method, PB B lineage cells could be divided into two populations according to their distinct repertoires of cell adhesion molecules and B cell antigens in untreated MM patients. The results show that: (i) the percentage and the absolute number of PB CD19⁺ B cells were decreased in MM patients compared with controls; (ii) the quantity and percentage of B cell antigens (CD20, CD22, CD24, DR, CD138) and adhesion molecules (β₁- and β₂-integrins, CD44, CD54, CD56, CD61 and CD62L) expressed by these PB CD19⁺ cells of MM patients and healthy subjects were similar and all of them were virtually polyclonal cells; (iii) a very minor circulating CD19^? CD38⁺⁺ CD45^?/dim subset was also detected which expressed CD138 (B-B4) (high intensity), monoclonal cytoplasmic immunoglobulin (cIg), and was negative for pan-B antigens (CD19, CD20, CD24, DR), surface immunoglobulin (sIg) and several adhesion molecules such as CD62L, CD18 and CD11a; this CD19^?CD38⁺⁺ CD45^?/dim CD138⁺⁺ subset was not found in normal blood and exhibited a phenotypic profile which was closely related to that of malignant BM plasma cells, with the exception of the CD56 antigen. Polymerase chain reaction (PCR) analysis of IgH clonotypic rearrangements confirmed these results. We postulate that, in MM patients, circulating B lineage cells may be divided into two different categories: polyclonal CD19⁺ B cells and a very minor proportion of clonal CD138⁺⁺ PC that escape from the BM.     

Amyloid deposition in primary pulmonary marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue

A rare association between primary pulmonary marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), and pulmonary immunoglobulin light chain (AL) amyloidosis is described in a 65-year-old woman suffering from rheumatoid arthritis (RA). All four nodules in the resected upper lobe of the lung had a similar histological appearance. They were composed of small-medium-sized atypical lymphocytes. Centrocyte-like cells had lymphoepithelial lesions. Immunohistochemically, the tumor cells clonally expressed B-cell markers, and demonstrated clonal rearrangement of the immunoglobulin heavy chain gene on polymerase chain reaction. Based on these findings the diagnosis of primary pulmonary MALT lymphoma was made. In addition, uniform eosinophilic material deposition was identified randomly within the tumor. It was Congophilic and exhibited apple-green birefringence on polarizing microscopy, and remained unaffected by potassium permanganate digestion. Deposited material was immunoreactive to lambda light chain. It was concluded that this material was AL amyloid in primary pulmonary MALT lymphoma. Plasma cells with mRNA of lambda chain was found infiltrated along the border of amyloid deposition. Finally, it is speculated that primary pulmonary MALT lymphoma developing in an autoimmune setting, RA in the present case, is associated with overproduction and abnormal clearance of immunoglobulin by the tumor cells, resulting in AL amyloidosis within the tumor.     

Restricted Expression of Immunoglobulin Light Chain mRNA and of the Adhesion Molecule CD11b on Circulating Monoclonal B Lineage Cells in Peripheral Blood of Myeloma Patients

Circulating monoclonal B cells in peripheral blood from patients with multiple myeloma or with monoclonal gammopathy of undetermined significance (MGUS) have previously been shown to express CD19, CD20, and PCA-1 and are predominantly CD45R0+, characterizing them as very late stage B cells. This work shows that the abnormal B cells are monoclonal as defined by their exclusive expression of either kappa or lambda light chain mRNA, and that the same type of light chain mRNA is expressed in both bone marrow plasma cells and blood B cells. These abnormal tumour-related circulating B cells express high densities of CD11b, a beta 2-integrin, which is expressed in a conformationally active state as defined by reactivity with monoclonal antibody 7E3. Normal peripheral blood B cells which do not bear CD11b acquire a high density after stimulation with pokeweed mitogen (PWM). At day 4 of culture, the expression of CD11b on normal CD19+ B cells was nearly comparable to that of the circulating myeloma late stage B cells. After PWM stimulation of circulating myeloma B cells the expression of CD11b was gradually lost during 4 days of culture, suggesting that its expression is dynamically regulated. Two patients with no phenotypically abnormal B cells in their blood at diagnosis acquired a large subset of CD11b+ B cells 4 weeks after initiation of chemotherapy. In most patients, a subset of the circulating myeloma B cells express a low density of CD5. The proportion of CD19+ B cells in the bone marrow expressing CD11b was much reduced compared with peripheral blood B cells, and CD11b was not detectable on plasma cells in the bone marrow, suggesting a sequential relationship of the B-cell subsets detected in our population of patients, involving gradual loss of CD11b concurrent with the loss of CD19 during B lineage differentiation. These cells appear to represent a continuously differentiating monoclonal B lineage culminating in the CD11b- plasma cell entrenched in the bone marrow. We speculate that the expression of conformationally active CD11b on the abnormal B cells in peripheral blood mononuclear cells of myeloma patients facilitates transendothelial migration of circulating myeloma B cells to the bone marrow.     

Anti-CD20 monoclonal antibody treatment of human herpesvirus 8-associated, body cavity-based lymphoma with an unusual phenotype in a human immunodeficiency virus-negative patient

Human herpesvirus 8 (HHV-8), or Kaposi's sarcoma-associated herpesvirus, is a gammaherpesvirus first detected in Kaposi's sarcoma tumor cells and subsequently in primary effusion lymphoma (PEL) tumor cells and peripheral blood mononuclear cells from PEL patients. PEL has been recognized as an individual nosologic entity based on its distinctive features and consistent association with HHV-8 infection. PEL is an unusual form of body cavity-based B-cell lymphoma (BCBL). It occurs predominantly in human immunodeficiency virus (HIV)-positive patients but occasionally also in elderly HIV-negative patients. We describe a case of PEL, with ascites, bilateral pleural effusions, and a small axillary lymphadenopathy, in a 72-year-old HIV-negative man. PCR performed on a lymph node specimen and in liquid effusion was positive for HHV-8 and negative for Epstein-Barr virus. The immunophenotype of the neoplastic cells was B CD19+ CD20+ CD22+ with coexpression of CD10 and CD23 and with clonal kappa light chain rearrangement. The patient was treated with Rituximab, a chimeric (human-mouse) anti-CD20 monoclonal antibody. Thirteen months later, the patient continued in clinical remission. This is the first report of an HHV-8-associated BCBL in an HIV-negative patient in Argentina.     

A comparison of morphologic features, flow cytometry, TCR-Vbeta analysis, and TCR-PCR in qualitative and quantitative assessment of peripheral blood involvement by Sézary syndrome

The strengths and weaknesses of various laboratory methods for peripheral blood (PB) Sézary cell quantitation have not been compared rigorously. In this study, manual Sézary cell counting, qualitative and quantitative flow cytometry, T-cell receptor (TCR) Vbeta flow cytometry, and TCR polymerase chain reaction were performed on PB specimens from 11 patients with Sézary syndrome (SS), 9 with reactive erythroderma, 6 with mycosis fungoides, and 11 healthy control subjects. These methods identified neoplastic cells in more than 90% of SS cases. The diagnostic specificities of these tests varied; they were enhanced by applying criteria proposed by the International Society for Cutaneous Lymphoma. Comparison of sequentially analyzed specimens from 6 patients with SS revealed that although the absolute number of clonal cells was reduced, in some cases, these cells still constituted the vast majority of the CD4+ T-cell subset, suggesting that quantitative subset analysis might be sufficient to monitor changes in the PB tumor burden.     

Clonal B cell expansions in patients with essential mixed cryoglobulinaemia.

In essential mixed, type II, cryoglobulinaemia (EMC) monoclonal autoantibodies with rheumatoid factor activity are synthesized at an accelerated rate by non-malignant B lymphocytes. In order to determine if the high cryoglobulin production rate is related to a clonal B cell expansion, cell surface markers of peripheral blood lymphocytes (PBL) were analysed by flow cytometry and the rearrangement of immunoglobulin (Ig) genes was investigated by Southern blot analysis of DNA extracted from the PBL of 12 EMC patients. Clonal expansion of B cells could be detected using DNA probes specific for the c kappa, c-mu, and JH genes in four out of 12 patients, two of whom also showed specific expansions of mu heavy and kappa light chain bearing cells using flow cytometry. The rearrangement of the c-myc locus was also noted in one of the patients with detectable Ig gene rearrangements. Demonstration of clonal B cell expansions in EMC patients shows that the clonal type of Ig gene rearrangements are not unique markers of malignant lymphomas but may also occur in autoimmune lymphoproliferative disorders. Since malignant B cell lymphomas can develop in a small number of EMC cases, the follow-up of these patients should be pursued indefinitely.     

Composite monoclonal B‐cell lymphocytosis and MYD88 L265P‐positive lymphoplasmacytic lymphoma in a patient with IgM light chain amyloidosis: Case report

Monoclonal B‐cell lymphocytosis (MBL) is an early or precursor asymptomatic proliferation of chronic lymphocytic lymphoma (CLL)‐like B‐cells. Lymphoplasmacytic lymphoma (LPL), often clinically associated with Waldenström macroglobulinemia, is a B‐cell neoplasm characterized by frequent MYD88 L265P mutation. Here, we report a rare composite MBL and LPL in a patient with IgM light chain (AL) amyloidosis. A 74‐year‐old male with a known IgM monoclonal protein developed proteinuria. No lymphocytosis was detected. Renal biopsy showed deposition of AL λ amyloid in the glomeruli and vessels. Subsequent bone marrow biopsy revealed nodular atypical CLL‐like small B‐cell proliferation and scattered peripheral LPL. Immunohistochemistry and/or flow cytometry revealed that the atypical CLL‐like population expressed CD19, CD20, CD5, weak CD23, LEF‐1 and diminished surface Igκ. The LPL was positive for CD19, CD20 and surface Igλ. Using laser‐capture microdissection and allele‐specific polymerase chain reaction, we confirmed that MYD88 L265P was detectable in the LPL but not in the atypical CLL‐like population. Thus, we demonstrated that these two populations were clonally independent, and made the diagnosis of composite MBL and LPL. An integrated clinical, pathological, immunophenotypic and genetic assessment is essential in such complicated cases, and especially ‘clone‐specific’ MYD88 genotyping may facilitate the differential diagnoses of low‐grade B‐cell lymphomas.     

T cell receptor diversity and activation markers in the V delta 1 subset of rheumatoid synovial fluid and peripheral blood T lymphocytes.

In the present study we have characterized the gamma/delta T cell receptor (TcR) population in synovial fluid (SF) and peripheral blood (PB) of patients with chronic inflammatory arthritis. By double staining we have shown that (a) synovial V delta 1+ cells have a high expression of activation markers CD45R0 ("memory cells") and HLA-DR as compared to PB, indicating a preactivated population of V delta 1-carrying T cells in vivo and (b) interleukin 2-induced expansion of synovial cells yields a high proportion of gamma/delta in most samples expressing predominantly the V delta 1 TcR. Junctional sequence analysis of the TcR delta chain from interleukin 2-expanded PB cell lines demonstrated a polyclonal V delta 1 population in three out of three samples. In SF cell lines three out of four samples were polyclonally expanded. In SF from one patient, however, a limited repertoire of expressed V delta 1 genes was found. Altogether, our data demonstrate the presence of preactivated V delta 1-expressing cells in the synovial compartment. This V delta 1 population is predominantly polyclonal, except in one patient where oligoclonally expanded V delta 1 cells were detected.     

Lack of deleterious somatic mutations in the CD95 gene of plasmablasts from systemic lupus erythematosus patients and autoantibody-producing cell lines

The interaction of CD95 with its ligand CD95L is important for negative selection of B cells during the germinal center (GC) reaction. Recently, mutations conferring resistance to CD95-induced apoptosis have been described for human GC B cells. Hence, as has been demonstrated for CD95-deficient mice, also GC-derived autoreactive B cells carrying somatic CD95 gene mutations may potentially service negative selection and participate in the development of autoimmune diseases. Here, single plasmablasts (PB) which are implicated in the production of autoantibodies in systemic lupus erythematosus (SLE) patients as well as ten human B cell lines producing autoantibodies were analyzed for destructive somatic CD95 gene mutations. However, inactivating CD95 gene mutations were very rare in PB and not detected in the cell lines. Sequence analysis of V gene rearrangements amplified from single PB confirmed that the cells are (post) GC B cells and additionally demonstrated massive clonal expansion of these cells in two of four SLE patients. We conclude that CD95 gene mutations play little if any role in the generation of the pool of PB in SLE patients and that mutations in the CD95 gene are rare among autoantibody-producing B cells in SLE and rheumatoid arthritis.     

Clonal Epstein-Barr virus genome in T-cell-rich lymphomas of B or probable B lineage.

Seventeen nodal lymphomas (originally diagnosed as T-cell lymphomas based on histological features and immunohistochemical staining results) were studied for the presence of Epstein-Barr virus (EBV) genome, and the results correlated with immunoglobulin and T-cell receptor gene rearrangement analyses performed on the same tissue samples. All four EBV positive cases had clonal rearrangement of the joining region of the immunoglobulin heavy chain (IgJH) gene without clonal T-cell receptor beta-chain (TCR beta) gene rearrangement. Of these, two cases also showed clonally rearranged light chain gene, and they were reclassified as T-cell rich B-cell lymphomas (TRBL). The other two cases lacked clonal kappa or lambda light chain rearrangement and they were reclassified as T-cell rich lymphomas of probable B lineage, based on their isolated IgJH clonal rearrangement. These B-cell lymphomas may be easily misdiagnosed as T-cell lymphomas owing to the presence of an abundant reactive T-cell infiltrate masking the tumor population. The florid T-cell reaction may represent an unusual host response towards a clonal proliferation of EBV bearing B cells.     

Analysis of VH genes rearranged by individual B cells in dermal infiltrates of patients with mycosis fungoides

In patients with cutaneous T cell lymphomas such as mycosis fungoides B cells can frequently be detected in the lymphocytic dermal infiltrate. To analyse their immunoglobulin heavy chain gene repertoire, single B cells were obtained from tissue sections of two typical patients with mycosis fungoides using hydraulic micromanipulation followed by specific amplification of the respective gene segments by single-cell polymerase chain reaction (PCR) technique. A total of 21 V_HDJ_H genes was sequenced. From each individual B cell a single productive V_HDJ_H rearrangement was obtained. There was no clonal relationship detected between any of these rearrangements suggesting polyclonality of the infiltrating B cells. The representation of V_H families was in accordance with the germ-line complexity. A remarkably high number of V_H genes (5/13 in patient 1; 3/8 in patient 2) was completely or nearly germ-line-identical. Five of seven V_H4 family genes were nearly unmutated. On the other hand, most of the V_H3 gene family members were somatically mutated in an antigen-driven manner. The proportion of germ-line-identical V_H genes, the usage of individual V_H, D, J_H gene elements, and the pattern of somatic mutations found in the B cells infiltrating skin lesions of patients with mycosis fungoides resembles the peripheral blood repertoire, suggesting a bystander role of these cells.     

The predictive value of bone marrow morphologic characteristics and immunostaining in primary (AL) amyloidosis.

The authors previously demonstrated that bone marrow plasmacytosis in primary (AL) amyloidosis may be monoclonal or polyclonal. However, the clinical implications of the degree of plasmacytosis and its clonality have not been studied. The authors evaluated 62 patients with AL amyloidosis, 40 of whom had monoclonal medullary plasma cells. There was complete concordance between the light chain class of the plasma cells in the monoclonal cases and that of the circulating paraprotein in the 22 cases associated with a paraprotein. The remaining 22 patients had polyclonal plasma cells, although a paraprotein was detected in 6. The degree of plasmacytosis was significantly higher among patients with monoclonal plasma cells and correlated inversely with length of survival. The authors' findings indicate that the quantitation of bone marrow plasma cells in AL amyloidosis by immunoperoxidase studies may predict the clinical course.     

Detection of clonal lambda light chain gene rearrangements in frozen and paraffin-embedded tissues by polymerase chain reaction.

A method was established to detect clonal lambda light chain gene rearrangements in peripheral blood lymphocytes and frozen or paraffin-embedded tissues. V lambda-gene-family-specific primers were used together with a J lambda primer mix in separate reactions to amplify V lambda gene rearrangements by the polymerase chain reaction. Clonal lambda gene rearrangements were detected in seven of seven lambda-expressing B cell leukemias, in four of five lambda-expressing non-Hodgkin's lymphomas with frozen tissues, and in seven of nine cases of lambda-expressing non-Hodgkin's lymphomas for which formalin-fixed, paraffin-embedded specimens were available. Clonality of amplified polymerase chain reaction products was confirmed by sequence analysis for several cases. The present study shows that it is possible to amplify clonal lambda gene rearrangements in the majority of lambda-expressing B cell leukemias and lymphomas. The method described here, therefore, is a useful supplement to the previously described approach of VH and VK gene amplification to detect clonal B cell populations and allows the study of V lambda gene usage and somatic mutation in lambda-expressing normal and malignant B cells.     

CD38, CD81 and BAFFR combined expression by transitional B cells distinguishes active from inactive systemic lupus erythematosus

In view of its heterogeneous presentation and unpredictable course, clinical management of systemic lupus erythematosus (SLE) is difficult. There is a need for biomarkers and diagnostic aids to monitor SLE disease activity and severity prior to, during and after treatment. We undertook this study to search for unique phenotypic patterns in each peripheral blood (PB) B cell subset, capable of distinguishing SLE patients with inactive disease versus SLE patients with active disease versus controls by using an automated population separator (APS) visualization strategy. PB was collected from 41 SLE patients and 28 age- and gender-matched controls. We analyzed the cell surface markers (in a tube CD20/CD27/CD19/CD45/CD38/CD81/BAFFR combination) expression on PB B cell subsets using principal component analysis, implemented in the APS software tool. Overall, our analysis indicates that active SLE can be distinguished from inactive SLE on the basis of a single tube analysis, focused on the decreased expression of CD38, CD81 and BAFFR in transitional B cells. The cluster analysis of immunophenotypic profiles of B cell subsets highlighted disease-specific abnormalities on transitional B cells that emerge as promising surrogate markers for disease activity. Further validation is needed with larger samples and prospective follow-up of patients.     

Autologous Mixed Lymphocyte Reactions in Patients with Rheumatoid Arthritis and Juvenile Rheumatoid Arthritis: Both Non-T Cells and in-Vivo-Activated T Cells Can Act as Stimulator Cells

The lymphocyte responses in autologous mixed lymphocyte reactions (AMLR) between irradiated non-T and T lymphocytes from the peripheral blood (PB) of rheumatoid arthritis (RA) and juvenile RA (JRA) patients were decreased compared with the AMLR responses of normal PB lymphocytes. Normal AMLR responses were seen in the synovial tissue and the synovial fluid lymphocytes from RA and JRA patients. The lymphocyte responses were also decreased in AMLR between irradiated non-T cells from peripheral blood and T cells from synovial tissue (ST) in RA patients and between irradiated non-T from PB and synovial fluid (SF) T cells in JRA patients. However, when irradiated non-T cells from ST of RA patients or from SF of JRA patients were mixed with autologous PB T lymphocytes, increased lymphocyte responses were observed. SF T lymphocyters and ST T cells were also shown to stimulate autologous PB T lymphocytes.     

Single cell analysis of B lymphocytes from Wegener's granulomatosis: B cell receptors display affinity maturation within the granulomatous lesions

Increased amounts of anti-neutrophil cytoplasm antibody (ANCA) directed against proteinase 3 (PR3) are a diagnostic and pathogenic hallmark of full-blown Wegener's granulomatosis (WG). Aggregates of B lymphocytes proximal to PR3+ cells as well as plasma cells have been described as substantial components of Wegener's granuloma and could participate in forming tertiary lymphoid structures, which might promote autoantibody formation. Our aim was a molecular analysis of single B cells in order to develop a methodological approach that allows examination of potential ANCA formation in the tissue. Single B cells from cryo-conserved endonasal biopsies of three WG patients were isolated, using laser-assisted microdissection. Subsequently, their immunoglobulin variable heavy (VH) and light (Vkappa, Vlambda) chain genes were analysed by single cell polymerase chain reaction and direct sequencing. Sixteen immunoglobulin VH-Vkappa or VH-Vlambda chain gene couples were characterized. Twelve of these immunoglobulin gene couples resembled memory B cells. Two offsprings of one B cell were detected, indicating clonal expansion. VH genes representing 39 single B cells of WG tissues displayed significantly more mutations when compared with VH genes from peripheral blood of a healthy donor. The findings confirm and extend our previous results, arguing for an initial selection and affinity maturation of B cells within Wegener's granuloma. Further, the methodology provides the initial basis for the recombinant generation of antibodies derived from tissue cells.