Variable region gene analysis of an isotype-switched (IgA) variant of chronic lymphocytic leukemia

Abstract Chronic lymphocytic leukemia of B-cell origin (B-CLL) is generally thought to arise by neoplastic transformation of B lymphocytes, which express CD5 and have features of an early stage of B-cell differentiation. To study isotype-switched B-CLL as a potentially more differentiated variant, we performed genetic and functional immunoglobulin (Ig) gene analysis in two cases of CD5+ B-CLL in which the peripheral blood mononuclear cells (PBMC) secreted predominantly IgA (CLL-249) or IgG (CLL-412) when stimulated with pokeweed mitogen in vitro. By cDNA sequencing and by studies of CLL-heterohybridomas, CLL- 249 expresses the heavy chain constant region C alpha as anticipated, while CLL-412 expresses C mu, not C gamma. In CLL-249, the expressed VH gene is 98% homologous to VH26, a germline VH3 gene that occurs frequently in the fetal repertoire, and which has been associated with anti-DNA specificity. The VL gene of CLL-249 is a lambda VL gene for which the germline sequence is not known. In CLL-412, the VH gene is 100% homologous to the VH1 gene of a published anti-DNA antibody (21/28), and is probably a germline gene sequence; the VL gene is 100% homologous to 15AVKI, also a germline gene. The supernatant antibody of the CLL-412 heterohybridoma is an IgM-kappa, which reacts with ssDNA and cardiolipin. The CLL-249 heterohybridoma secreted IgA-lambda, which bound none of the antigens tested, a finding that may be related to amino acid differences from the probable germline V genes. The demonstration of an in vivo isotype-switched variant, such as CLL-249, suggests that B-CLL may be a heterogeneous group of clonal disorders, of which less common variants may have features of more differentiated B-cell stages, such as isotype switching.

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Variable region gene analysis of an isotype-switched (IgA) variant of chronic lymphocytic leukemia

Blood ◽

10.1182/blood.v80.9.2287.bloodjournal8092287 ◽

1992 ◽

Vol 80 (9) ◽

pp. 2287-2297 ◽

Cited By ~ 1

Author(s):

DF Friedman ◽

JS Moore ◽

J Erikson ◽

J Manz ◽

J Goldman ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Mononuclear Cells ◽

Early Stage ◽

Lymphocytic Leukemia ◽

Gene Analysis ◽

Pokeweed Mitogen ◽

B Cell Differentiation ◽

Germline Gene ◽

Vh Gene

Chronic lymphocytic leukemia of B-cell origin (B-CLL) is generally thought to arise by neoplastic transformation of B lymphocytes, which express CD5 and have features of an early stage of B-cell differentiation. To study isotype-switched B-CLL as a potentially more differentiated variant, we performed genetic and functional immunoglobulin (Ig) gene analysis in two cases of CD5+ B-CLL in which the peripheral blood mononuclear cells (PBMC) secreted predominantly IgA (CLL-249) or IgG (CLL-412) when stimulated with pokeweed mitogen in vitro. By cDNA sequencing and by studies of CLL-heterohybridomas, CLL- 249 expresses the heavy chain constant region C alpha as anticipated, while CLL-412 expresses C mu, not C gamma. In CLL-249, the expressed VH gene is 98% homologous to VH26, a germline VH3 gene that occurs frequently in the fetal repertoire, and which has been associated with anti-DNA specificity. The VL gene of CLL-249 is a lambda VL gene for which the germline sequence is not known. In CLL-412, the VH gene is 100% homologous to the VH1 gene of a published anti-DNA antibody (21/28), and is probably a germline gene sequence; the VL gene is 100% homologous to 15AVKI, also a germline gene. The supernatant antibody of the CLL-412 heterohybridoma is an IgM-kappa, which reacts with ssDNA and cardiolipin. The CLL-249 heterohybridoma secreted IgA-lambda, which bound none of the antigens tested, a finding that may be related to amino acid differences from the probable germline V genes. The demonstration of an in vivo isotype-switched variant, such as CLL-249, suggests that B-CLL may be a heterogeneous group of clonal disorders, of which less common variants may have features of more differentiated B-cell stages, such as isotype switching.

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The B cell IgM Fc receptor: further evidence for the B cell origin of "null" chronic lymphocytic leukemia

Blood ◽

10.1182/blood.v64.2.375.bloodjournal642375 ◽

1984 ◽

Vol 64 (2) ◽

pp. 375-379

Author(s):

RA Rudders ◽

PA Poldre

Keyword(s):

Cell Differentiation ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Surface Membrane ◽

Fc Receptor ◽

Lymphocytic Leukemia ◽

B Cell Differentiation ◽

Facs Analysis ◽

Membrane Expression ◽

Surface Immunoglobulin

Fifty-three cases of chronic lymphocytic leukemia (CLL) were studied for the presence of the B cell IgM Fc receptor (Fc microR) using an aggregated IgM reagent. Restricted surface immunoglobulin, using conventional immunofluorescent techniques and FACS analysis, was detected in 43 cases (81%). The cells in the remaining ten cases (19%) expressed negligible surface immunoglobulin (slg-) and did not form E rosettes (E-), but this “null” subset clearly expressed the B cell Fc microR. The coincident membrane expression of the B1 antigen and the la- like antigen, as well as serial studies showing surface membrane light chain acquisition (in one patient), provided additional evidence for the B cell origin of this slg-E- subset. This subgroup of CLL appears to correspond phenotypically to a normal counterpart at a stage of B cell differentiation between the pre-B cell and the slgM+ early B cell. The B cell Fc microR appears to be a consistent and potentially useful marker for sl gE (“null”) CLL.

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Prognostic importance of serum soluble CD23 level in chronic lymphocytic leukemia

Blood ◽

10.1182/blood.v88.11.4259.4259 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4259-4264 ◽

Cited By ~ 116

Author(s):

M Sarfati ◽

S Chevret ◽

C Chastang ◽

G Biron ◽

P Stryckmans ◽

...

Keyword(s):

Overall Survival ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Disease Progression ◽

Early Stage ◽

Lymphocytic Leukemia ◽

Study Entry ◽

Clinical Staging ◽

Soluble Cd23

Abstract Prognosis of B-cell chronic lymphocytic leukemia (CLL) is based on clinical staging whose limitation is the failure to assess whether the disease will progress or remain stable in early stage (Binet A, or Rai 0, I, II) patients. We previously reported that soluble CD23 (sCD23), a protein derived from the B-cell membrane CD23 Ag, is selectively elevated in the serum of CLL patients. This prospective study assessed the predictive value of serum sCD23 level measured at study entry on the overall survival of all CLL patients and on disease progression of stage Binet A patients. Prognostic value of repeated measurements of sCD23 over time in stage A patients was also analyzed. One hundred fifty-three CLL patients were prospectively followed with a median follow-up of 78 months. Eight clinical or biological parameters were collected from the date of the first sCD23 measurement. At study entry, by Cox model, Binet staging (P = .0001) and serum sCD23 level (P = .03) appeared as prognostic factors for survival. Patients with sCD23 level above median value (> 574 U/mL) had a significantly worse prognosis than those with lower values (median survival of 53 v 100+ months, P = .0001). During follow-up, sCD23 doubling time increased by 3.2 the risk of death (P = .001). Among stage A patients (n = 100), sCD23 determination at study entry was the sole variable predictive of disease progression, patients with sCD23 level above 574 U/mL had a median time progression of 42 months versus 88 months for those with lower levels (P = .0001). Stage A patients who doubled their sCD23 level exhibited a 15-fold increased risk of progression (P = .0001) and, in addition, the sCD23 increase preceded by 48 months disease progression. We conclude that in CLL patients, serum sCD23 level provides significant additional prognostic information in terms of overall survival. Most interestingly, among early stage patients, sCD23 determination at diagnosis and during the course of the disease may help to the early identification of patients who will rapidly progress to upper stages.

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Serum Levels of Soluble CD23, but not Soluble CD25, Predict Disease Progression in Early Stage B-Cell Chronic Lymphocytic Leukemia

Leukemia & Lymphoma ◽

10.3109/10428199709058320 ◽

1997 ◽

Vol 27 (5-6) ◽

pp. 523-532 ◽

Cited By ~ 30

Author(s):

W. U. Knauf ◽

I. Langenmayer ◽

B. Ehlers ◽

B. Mohr ◽

D. Adorf ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Disease Progression ◽

Early Stage ◽

Serum Levels ◽

Lymphocytic Leukemia ◽

Soluble Cd25 ◽

Soluble Cd23 ◽

Cell Chronic Lymphocytic Leukemia ◽

Predict Disease Progression

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Evidence for progenitors of chronic lymphocytic leukemia B cells that undergo intraclonal differentiation and diversification

Blood ◽

10.1182/blood.v87.4.1586.bloodjournal8741586 ◽

1996 ◽

Vol 87 (4) ◽

pp. 1586-1594 ◽

Cited By ~ 27

Author(s):

M Dono ◽

S Hashimoto ◽

F Fais ◽

V Trejo ◽

SL Allen ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Dna Sequences ◽

Mononuclear Cells ◽

Lymphocytic Leukemia ◽

Specific Primers ◽

Region Gene ◽

Peripheral Blood Mononuclear ◽

Ancestral Gene

Peripheral blood mononuclear cells from five patients with IgG+ B-type chronic lymphocytic leukemia (B-CLL) were analyzed for the presence of clone-specific Ig H chain variable region gene mRNA transcripts linked to C mu and/or C alpha. This was assessed by (1) comparing the lengths of portions of the VHDJH of the IgG+ CLL clones with those of the mu and alpha isotype-expressing B cells, (2) performing clone-specific endonuclease digestion studies, and (3) determining the DNA sequences of the mu and alpha isotype-expressing cDNA. Thus, when B-cell mRNA from these five patients were reverse transcribed with C gamma-specific primers and then amplified by polymerase chain reaction, dominant cDNA were found with lengths corresponding to those of the IgG+ CLL B cell. In addition, in four cases, cDNA of lengths identical to those of the CLL B cell were detected when mRNA was reverse transcribed and amplified using c mu- and/or C alpha-specific primers, strongly suggesting clonal relatedness. These CLL-related mu- and alpha- expressing cDNA were present in greater amounts that unrelated (non- CLL) mu- and alpha-expressing cDNA from normal B cells that used genes of the same VH family. When the sequences of these CLL-related C mu- and C alpha-expressing cDNA were compared with those of the IgG+ CLL clones, it was clear that they were derived from the same ancestral gene as the IgG-expressing CLL B cell, thus documenting their common origin. Finally, nucleotide point mutations were observed in the mu- and alpha-expressing cDNA of certain patients, indicating divergence with the CLL. These data suggest that IgM+ B cells, which are precursors of the leukemic B cells, exist in increased numbers in the blood of most patients with IgG+ B-CELL and that these cells may differentiate, accumulate V genes mutations, and undergo isotype switching in vivo. In addition, the data are consistent with a sequential-hit model for the evolution of CLL.

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The Receptor for Hyaluronic Acid Mediated Motility (RHAMM/CD168) Is a Potential Target for Immunotherapy of Patients with B-Cell Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v106.11.53.53 ◽

2005 ◽

Vol 106 (11) ◽

pp. 53-53 ◽

Cited By ~ 1

Author(s):

Krzysztof Giannopoulos ◽

Iwona Hus ◽

Li Li ◽

Agnieszka Bojarska-Junak ◽

Jochen Greiner ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Mononuclear Cells ◽

Tissue Expression ◽

T Cell Response ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Peripheral Blood Mononuclear ◽

Cell Chronic Lymphocytic Leukemia

Abstract Definition of appropriate target antigens might open new avenues to antigen targeted immunotherapies for patients with B-cell chronic lymphocytic leukemia (B-CLL). We screened the mRNA expression of tumor associated antigens (TAAs), from the literature (fibromodulin, survivin, OFA-iLRP, BAGE, G250, MAGE1, PRAME, proteinase, Syntaxin, hTERT, WT-1), and TAAs defined earlier by serological analysis of cDNA expression libraries from leukemic cells (PINCH, HSJ2, MAZ, MPP11, RHAMM/CD168, NY-Ren60). Peripheral blood mononuclear cells from 43 B-CLL patients and 20 healthy volunteers (HVs) were examined by conventional and quantitative RT-PCR. mRNA of RHAMM/CD168, fibromodulin, syntaxin and NY-Ren60 was expressed in 55–90%, mRNA of HSJ2, MAZ and OFA-iLRP in 90–100% of the patients. No expression of WT-1, h-TERT, BAGE, G250, MAGE1 and survivin was observed. Low (2–20%) expression frequencies of MPP11, PINCH, PRAME and proteinase were detected. RHAMM/CD168, fibromodulin, PRAME and MPP11 showed expression in B-CLL patients, but not in HVs. Because of the exquisite tissue expression of RHAMM/CD168 and its high expression frequency in B-CLL patients even in early stages of disease, mixed lymphocyte peptide culture (MLPC), enzyme-linked immunosorbent spot (ELISPOT) and flow cytometry were performed for antigen specific T cells. In MLPC, RHAMM specific responses by CD8+HLA-A2/R3tetramer+CCR7-CD45RAhigh effector T cells were detected. Moreover, we observed an enhanced RHAMM/CD168 specific CD8+ T cell response after vaccination in one from four HLA-A2 positive B-CLL patients vaccinated with tumor cell lysate pulsed dendritic cells. Therefore, RHAMM/CD168 is an interesting candidate antigen for future immunotherapies in both ZAP-70 (+) and ZAP-70 (−) B-CLL patients.

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RHAMM/CD168 Is a Novel Leukemia Associated Antigen with Prognostic Value for Patients with B-Cell Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v108.11.2773.2773 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2773-2773

Author(s):

Krzysztof Giannopoulos ◽

Alexander Krober ◽

Anna Dmoszynska ◽

Jacek Rolinski ◽

Hartmut Dohner ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cd8 T Cells ◽

Mononuclear Cells ◽

Surrogate Marker ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Free Survival ◽

Cell Chronic Lymphocytic Leukemia

Abstract Background and Aims: Differential expression of molecules in patients with B-cell chronic lymphocytic leukemia (B-CLL) might define suitable targets for T cell based vaccines and/or antibody approaches. Methods: We assessed the mRNA expression of the tumor associated antigen (TAA) RHAMM/CD168 defined earlier by serological analysis of cDNA expression libraries (SEREX) from leukemic cells. Results: Peripheral blood mononuclear cells from 40 B-CLL patients and 20 healthy volunteers (HVs) were examined by quantitative RT-PCR. A leukemia-restricted expression of the antigen RHAMM/CD168 was observed in 39/40 B-CLL patients in contrast to the absence of its expression in HVs. To evaluate the immunogenicity of this novel LAA, mixed lymphocyte peptide cultures (MLPCs), followed by enzyme-linked immunosorbent spot (ELISPOT) and flow cytometry assays were performed to detect antigen-specific CD8+ T cells. RHAMM/CD168 specific responses by CD8+ HLA-A2/R3tetramer+CCR7-CD45RAhigh effector T cells were detected. As these CD8+ T cells contribute to the elimination of RHAMM+ CLL cells, we questioned whether expression of the antigen would be associated with a better survival. RHAMM/CD168 expression revealed to be higher in patients with unmutated IgVH status. RHAMM normalized against the housekeeping gene TATA binding protein (TBP), i.e. the RHAMM/TBP ratio was defined as a prognostic surrogate marker for B-CLL. B-CLL patients with a RHAMM/TBP ratio > 1.67 showed a significantly shorter treatment free survival (TFS) (Fig. 1). A tendency towards higher RHAMM/TBP expression ratios was observed in B-CLL cases with del11q. Conclusion: RHAMM/CD168 is a novel LAA in B-CLL patients, an antigen correlating with the clinical course of the disease. Therefore, we consider RHAMM/CD168 an interesting target for immunotherapy in early stage B-CLL patients, especially with worse prognosis (IgVH unmutated). Figure 1. Treatment-free-survival (TFS) according to RHAMM/TBP ratio Figure 1. Treatment-free-survival (TFS) according to RHAMM/TBP ratio

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Tyro3, Axl Ve Mer (TAM) Receptors On Surfaces of Mononuclear Cells in Patients with B-Cell Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v120.21.4588.4588 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4588-4588

Author(s):

Dilvin Guney ◽

Aysin Tulunay ◽

Funda Pepedil ◽

Isik Kaygusuz ◽

Cafer Adiguzel ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Gradient Centrifugation ◽

Lymphocytic Leukemia ◽

Control Group ◽

Ligand Molecule ◽

Tam Receptors ◽

Cell Chronic Lymphocytic Leukemia

Abstract Abstract 4588 Background: Tyro 3 (Sky), Axl, and Mer receptors are members of the family of tyrosine kinases and Gas6 is their ligand molecule. In some types of cancer, upregulation of Axl/Gas6 indicated a worse prognosis, but an opposite situation was observed in renal “cell” carcinoma. This contradiction may suggest that Axl/Gas6 pathway varies depending on the type of cancer. The objective of this study is to investigate TAM receptors on surfaces of mononuclear cells in patients with B-Cell chronic lymphocytic leukemia (B-Cell-CLL). Material & Methods: B-Cell-CLL patients (grade 0–1, according to the classification of RAI), who were not on a drug treatment, were recruited in this study (n= 20; 9 female, 11 male). Their ages were 44 to 74 (mean: 63), and the control group consisted of 13 healthy volunteers (5 female, 8 male), whose age range is 20–89 (mean: 36). Mononuclear cells were isolated by density gradient centrifugation, and then surface TAM receptors were detected by flow cytometry. Mononuclear cell were stained with the primary antibodies against Tyro3, Axl and Mer. Results: The percentage of the surface TAM receptors on mononuclear cells from the patient group (25–75% interquartile range): Tyro 3= 25.50 (4.2– 45.62); Axl= 17/55 (5.57– 36.32), and Mer= 19.90 (1.92– 37.55). In the control group the following values were obtained: Tyro 3= 2.60 (1.35–3.25); Axl= 0.9 (0.4–2.6), and Mer= 2.50 (0.35–3.65). The percentage of three of them was significantly higher in the B-Cell-CLL group than those in the control group (P<0.01). Conclusion: In conclusion, this preliminary study showed that TAM receptors on surfaces of mononuclear cells are higher in patients with B-Cell-CLL patients than the control group. Gas6/TAM signaling may play a potential role in the pathogenesis of B Cell-CLL. Further studies are required to elucidate the actual role of Gas6/TAM signaling in B-Cell-CLL. Gas6/TAM signaling might be a new strategic goal for the treatment of B-Cell-CLL. Disclosures: No relevant conflicts of interest to declare.

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