Emergence of a B-cell lymphoblastic lymphoma in a patient with B-cell chronic lymphocytic leukemia: evidence for the single-cell origin of the two tumors

A patient is described who presented with a chronic lymphocytic leukemia (CLL) and later developed a lymphoblastic lymphoma. The cells from the CLL were typical mature B lymphocytes as could be assessed by morphologic, cytochemical, and surface marker analyses. The cells from the lymphoblastic lymphoma were immature B cells that expressed CD10, CD20, and HLA-DR markers, but not surface Ig or cytoplasmic mu chains, and were negative for terminal deoxynucleotidyl transferase (TdT). The cells of two continuous cell lines, obtained from the bone marrow and the peripheral blood of the patient, had the same phenotype as the lymphoblastic lymphoma cells, did not contain the Epstein-Barr virus genome, and displayed malignant features in vitro, including the capacity to form colonies in agar. The two cell lines also shared identical chromosomal abnormalities, a finding which suggests that they derived from the same malignant cell already present in vivo. Such chromosomal abnormalities were not seen in the karyotype of the peripheral blood cells at the onset of the disease. Analysis of the Ig heavy chain genes using a DJ-specific probe showed the very same monoclonal rearrangement in the cells from the B-CLL, the lymphoblastic lymphoma and the two cell lines, thus demonstrating their common clonal origin. By contrast, a monoclonal rearrangement of the lambda chain gene locus was found in the B-CLL cells only, a finding consistent with their exclusive capacity to express surface IgM lambda. This patient represents a rare case in whom a chronic lymphoproliferative disorder with mature malignant cells transforms into a lymphoblastic lymphoma characterized by cells frozen at a very early maturational stage. The possible mechanisms leading to such transformation within the same cell clone are discussed.

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Emergence of a B-cell lymphoblastic lymphoma in a patient with B-cell chronic lymphocytic leukemia: evidence for the single-cell origin of the two tumors

Blood ◽

10.1182/blood.v78.3.797.797 ◽

1991 ◽

Vol 78 (3) ◽

pp. 797-804 ◽

Cited By ~ 16

Author(s):

V Pistoia ◽

S Roncella ◽

PF Di Celle ◽

M Sessarego ◽

G Cutrona ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Lines ◽

Peripheral Blood ◽

Chromosomal Abnormalities ◽

Cell Clone ◽

Lymphoblastic Lymphoma ◽

Lymphocytic Leukemia ◽

Terminal Deoxynucleotidyl Transferase ◽

Chain Gene

Abstract A patient is described who presented with a chronic lymphocytic leukemia (CLL) and later developed a lymphoblastic lymphoma. The cells from the CLL were typical mature B lymphocytes as could be assessed by morphologic, cytochemical, and surface marker analyses. The cells from the lymphoblastic lymphoma were immature B cells that expressed CD10, CD20, and HLA-DR markers, but not surface Ig or cytoplasmic mu chains, and were negative for terminal deoxynucleotidyl transferase (TdT). The cells of two continuous cell lines, obtained from the bone marrow and the peripheral blood of the patient, had the same phenotype as the lymphoblastic lymphoma cells, did not contain the Epstein-Barr virus genome, and displayed malignant features in vitro, including the capacity to form colonies in agar. The two cell lines also shared identical chromosomal abnormalities, a finding which suggests that they derived from the same malignant cell already present in vivo. Such chromosomal abnormalities were not seen in the karyotype of the peripheral blood cells at the onset of the disease. Analysis of the Ig heavy chain genes using a DJ-specific probe showed the very same monoclonal rearrangement in the cells from the B-CLL, the lymphoblastic lymphoma and the two cell lines, thus demonstrating their common clonal origin. By contrast, a monoclonal rearrangement of the lambda chain gene locus was found in the B-CLL cells only, a finding consistent with their exclusive capacity to express surface IgM lambda. This patient represents a rare case in whom a chronic lymphoproliferative disorder with mature malignant cells transforms into a lymphoblastic lymphoma characterized by cells frozen at a very early maturational stage. The possible mechanisms leading to such transformation within the same cell clone are discussed.

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Contribution of Comparative Genomic Hybridization and Fluorescence in situ Hybridization to the Detection of Chromosomal Abnormalities in B-Cell Chronic Lymphocytic Leukemia

Oncology Research and Treatment ◽

10.1159/000050284 ◽

2001 ◽

Vol 24 (1) ◽

pp. 60-65 ◽

Cited By ~ 2

Author(s):

M. Jarošová ◽

K. Jedličková ◽

M. Holzerová ◽

R. Urbanová ◽

T. Papajík ◽

...

Keyword(s):

In Situ Hybridization ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Fluorescence In Situ Hybridization ◽

Comparative Genomic Hybridization ◽

Chromosomal Abnormalities ◽

Lymphocytic Leukemia ◽

Comparative Genomic ◽

Cell Chronic Lymphocytic Leukemia

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Sphingosine 1-Phosphate (S1P) Induces Migration and ERK/MAP-Kinase-Dependent Proliferation in Chronic Lymphocytic Leukemia (B-CLL) Due to Expression of the G Protein-Coupled Receptors S1P1/4.

Blood ◽

10.1182/blood.v106.11.4996.4996 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4996-4996

Author(s):

Gabriele Seitz ◽

Sedat Yildirim ◽

Andreas M. Boehmler ◽

Lothar Kanz ◽

Robert Möhle

Keyword(s):

Chronic Lymphocytic Leukemia ◽

G Protein ◽

Cell Lines ◽

Peripheral Blood ◽

G Protein Coupled Receptors ◽

Lymphocytic Leukemia ◽

S1p Receptors ◽

Sphingosine 1 Phosphate ◽

G Protein Coupled

Abstract Egress of lymphocytes from lymphoid organs into the circulation has been shown to depend on the presence of the lipid mediator sphingosine 1-phosphate (S1P) in the peripheral blood, and expression of corresponding S1P receptors (i.e., S1P1), that belong to the family of 7-transmembrane G protein-coupled receptors (GPCR). As circulating lymphocytic lymphoma cells are a hallmark of chronic lymphocytic leukemia, we analyzed expression of different S1P receptors and the effects of S1P on B-CLL cells. By qualitative and quantitative (TaqMan) RT-PCR, significant mRNA expression of S1P1 and S1P4 was found in CLL cell lines (EHEB, MEC-1) and in most samples (S1P1 in 88%, S1P4 in 100%) of primary CD19+ cells isolated from the peripheral blood of untreated B-CLL patients. mRNA of other S1P receptors (S1P2, S1P3, S1P5) was less consistently detected. Normal, nonmalignant B cells were strongly positive for S1P1, while other S1P receptors were weakly expressed or negative. S1P induced typical effects of chemotactic GPCR, such as actin polymerization (analyzed by flow cytometry) and chemotaxis (measured in a modified Boyden chamber assay) in CLL cell lines and primary B-CLL cells. After serum deprivation in vitro, S1P induced phosphorylation of ERK/MAP-kinase as analyzed by Western blot, demonstrating that S1P receptors expressed in CLL were able to activate signaling pathways of GPCR not only related to cell migration and chemotaxis, but also to cell proliferation. Of note, the S1P1 ligand FTY720, which induces receptor internalization after prolonged exposure and acts as an antagonist, resulted in apoptosis in CLL cell lines and primary CLL cells in vitro, as measured by MTT-test and staining with Annexin-FITC, respectively. We conclude that sphingosine 1-phosphate, which is present in the peripheral blood in considerable amounts, contributes to the trafficking of B-CLL cells expressing the GPCRs S1P1/4, and to their prolonged survival.

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Influence of Chromosomal Aberrations on Response to First Line Therapy in B-Cell Chronic Lymphocytic Leukemia: Interim Analysis of PALG-CLL3 Randomized Study Comparing Cladribine Plus Cyclophosphamide vs Fludarabine Plus Cyclophosphamide.

Blood ◽

10.1182/blood.v110.11.2046.2046 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2046-2046

Author(s):

Tadeusz Robak ◽

Jerzy Z. Blonski ◽

Ewa Wawrzyniak ◽

Aleksandra Palacz ◽

Joanna Gora-Tybor ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Chromosomal Aberrations ◽

Interim Analysis ◽

Chromosomal Abnormalities ◽

Complete Response ◽

Lymphocytic Leukemia ◽

Lower Probability ◽

Trisomy 12 ◽

Cell Chronic Lymphocytic Leukemia

Abstract Impact of cytogenetic abnormalities on treatment with different purine nucleoside analogs in patients (pts) with B-cell chronic lymphocytic leukemia (B-CLL) is largely unknown. One of objectives of PALG-CLL3 trial, comparing cladribine plus cyclophosphamide (CC) with fludarabine plus cyclophosphamide (FC) in previously untreated progressive B-CLL, was to verify the response to treatment in subsets of pts characterized by common cytogenetic abberrations. Chromosomal abnormalities were assessed using fluorescence in situ hybridization (FISH) on interphase nuclei of lymphocytes on whole blood smears prior to the start of the study treatment. Pts were screened for trisomy 12, deletions (del) 11q, del 13q and del 17p using DNA probes: CEP12, LSI: ATM, D13S319 and p53 (Vysis), respectively. For the purpose of the present interim analysis complete cytogenetic results were available in 133 pts out of 423 pts included to the study. In this group the chromosomal aberrations were detected in 102 pts (77%) including single abnormalities observed in 69 pts (52%) and two or more aberrations in 33 pts (25%). Thirty-one pts (23%) exhibited a normal interphase FISH pattern. The most frequent single abnormality was del 13q found in 38 pts (29%), while del 17p, trisomy 12 and del 11q were identified in 14 pts (11%), 11 pts (8%), and 6 pts, (5%), respectively. The most frequently observed associations of chromosomal aberrations were: del 13q with del 11q (11 pts, 8%) and del 13q with del 17p (10 pts, 8%). Four pts (3%) revealed three chromosomal abnormalities including association of trisomy 12/del 11q/del 13q in two pts, trisomy 12/del 11q/del 17p in one pt and del 11q/del 13q/del 17p in one pt. Overall, treatment was completed and response assessed in 113 out of 133 pts with known FISH pattern. In this group of pts del 17p was the only chromosomal abnormality that correlated significantly with treatment outcome. Pts with del 17p (21, 19%) had lower probability to achieve a complete response (CR) (0.044). Interestingly, in independent analyses of both treatment arms, the negative impact of 17p was seen in pts treated with FC (p=0.002), but not in pts treated with CC (p=0.6). Moreover, comparing response rates between treatment arms we found that CC was superior to FC in terms of complete response in pts with del 17p (57% CR in CC v 14% CR in FC arm, p=0.04). In conclusion, chromosomal abnormalities can be detected in majority B-CLL pts requiring treatment. Our preliminary results suggest that CC combination may have some advantage in terms of CR achievement in B-CLL pts harboring del 17p.

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OSU-DY7, a Novel D-Tyrosinol Derivative, Mediates Cytotoxicity in Chronic Lymphocytic Leukemia and Lymphoblastic Lymphoma through p38 Mitogen-Activated Protein Kinase Pathway.

Blood ◽

10.1182/blood.v114.22.3778.3778 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3778-3778

Author(s):

Li-Yuan Bai ◽

Yihui Ma ◽

Samuel K Kulp ◽

Shu-Huei Wang ◽

Chang-Fang Chiu ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

P38 Mapk ◽

Cell Lines ◽

Alternative Therapy ◽

Lymphoblastic Lymphoma ◽

Lymphocytic Leukemia ◽

Parp Cleavage ◽

Raji Cells ◽

Lymphocytic Cell

Abstract Abstract 3778 Poster Board III-714 Introduction Chronic lymphocytic leukemia (CLL) is a most common form of adult leukemia with minimal treatment options. Drug resistance and associated immune deregulation limit use of current therapies, thus warranting need for alternative therapy development. Our laboratories recently identified a novel D-tyrosinol-derived compound targeting p38 MAPK pathway, OSU-DY7 [(R)-2-amino-3-(4-heptyloxy-phenyl)-propan-1-ol]. Here we demonstrate the efficacy of OSU-DY7 for lymphocytic cell lines and primary B cells from CLL patients. Materials and Methods We tested the pre-clinical efficacy of OSU-DY7 in primary CLL B cells and B cell lines representing CLL (MEC-1), ALL (697), and lymphoblastic lymphoma (Raji and Ramos). Results The cytotoxicity of OSU-DY7 was both dose- and time-dependent. The IC50 of OSU-DY7 at 24 hrs for MEC-1, 697, Raji, Ramos and primary B-CLL cells were 2.40 μM, 10.39 μM, 6.04 μM, 3.75 μM and 3.58 μM respectively. OSU-DY7 induced activation of caspase-3 and poly (adenocine diphosphate-ribose) polymerase (PARP) cleavage. Pancaspase inhibitor Z-VAD-FMK partially rescued OSU-DY7-induced cytotoxicity in CLL B cells and cell lines. Interestingly, OSU-DY7 mediated-cytotoxicity was associated with increased phosphorylation of p38 MAPK and its downstream target protein MAPKAPK2 in both lymphocytic cell lines and primary B-CLL cells. Compared with control group, the ratio of p-p38MAPK (Thr180Tyr182) versus p38MAPK in Raji cells treated with 2 μM, 4 μM and 8 μM of OSU-DY7 increased 2.2, 4.7 and 11 fold respectively (p<0.0001). Similar increase in p38MAPK phosphorylation was also observed in six CLL patient samples after treatment with OSU-DY7 2 μM, 4 μM and 8 μM for 24 hours (p=0.0004). Moreover, SB202190, a specific p38 MAPK inhibitor, decreased MAPKAPK2 protein level with concomitant rescue of the cells from the OSU-DY7 mediated-cytotoxicity. Thus pretreatment of Raji and primary CLL B cells with SB202190 reduced the OSU-DY7 induced cytotoxicity by 36.7% and 24.3% respectively by 24hrs (Raji:p<0.0001; primary CLL B cells: p<0.005 ). Furthermore, SB202190 rescued OSU-DY7 down regulated survivin protein by ∼2 fold (p<0.0001, n=3) and mRNA levels by 2.4 fold (95% CI: 1.56∼3.84 fold, p=0.0026, n=3) in Raji cells indicating a role for p38MAPK dependent regulation of survivin by OSU-DY7. Conclusions This study provides an evidence for a role of OSU-DY7 in p38 MAPK dependent activation and survivin down regulation associated with apoptosis in lymphocytic cells, thus warranting further development of this novel agent for alternative therapy for lymphocytic malignancies. [This work was supported by D. Warren Brown Foundation and Leukemia and Lymphoma Society] Disclosures: No relevant conflicts of interest to declare.

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Comparison of Cytotoxic Mechanisms of Anti-CD20 Antibodies GA101 and Rituximab Against Fresh Chronic Lymphocytic Leukemia Cells

Blood ◽

10.1182/blood.v116.21.2467.2467 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2467-2467

Author(s):

Lina Reslan ◽

Stéphane Dalle ◽

Cindy Tournebize ◽

Stephanie Herveau ◽

Emeline Cros ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Cell Death ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Peripheral Blood ◽

Lymphocytic Leukemia ◽

Oxygen Species ◽

Cytochrome C Release ◽

Anti Cd20 ◽

Cd20 Antibodies

Abstract Abstract 2467 GA101, a novel glycoengineered type II IgG1 antibody against CD20, has shown a direct and immune effector cell-mediated cytotoxicity in numerous B-cell disorders. Chronic Lymphocytic Leukemia (CLL) is the most common hematologic malignancy in the western world. Since circulating mature B-CLL cells express high levels of antiapoptotic proteins that are implicated in the survival mechanism, we investigated whether the effects of GA101 compared to rituximab, induces apoptosis in these cells and what mechanism underlies GA101-mediated cytotoxicity. CLL cells were isolated from peripheral blood samples by density gradient centrifugation and B lymphocytes were purified by a negative selection method using the EasySep® B Cell Enrichment Cocktail. Cell viability was measured flow cytometrically by annexinV binding. We assessed the mitochondrial transmembrane potential (ΔΨm) by staining with 3,3-dihexyloxacarbocyanine iodide (DiOC6[3]), the generation of reactive oxygen species by staining with Dihydroethidine (DHE) as well as cytochrome c release. Moreover, the expression of several apoptosis-regulating proteins, including the Bcl-2 family proteins (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak and Bad) and the activation of the caspase cascade were evaluated by immunoblotting on 34 fresh peripheral blood B-CLL specimens. We showed that GA101 initiates an early extensive cell death. The average decrease of viability of freshly isolated and purified CLL cells 24 hours post-treatment with 10μg/ml of anti CD20 antibodies were 37.6% for GA101 (n=11) and 28.8% for Rituximab (n=11). The GA101-induced cell death was paralleled by a rapid loss of mitochondrial membrane potential accompanied with the production of ROS and cytochrome c release that occurred significantly as early as 3 hours post-treatment. However, rituximab was unable to initiate a loss of ΔΨm and the production of ROS. The use of antioxidants such as N-acetyl cysteine and L-ascorbic acid were unable to circumvent either the GA101-induced cell death or the loss of ΔΨm. However, the preincubation of CLL cells with Z-VAD.fmk (N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone), a broad caspase inhibitor, abolished the exposure of phosphatidylserine residues, the generation of reactive oxygen species and reversed the loss of ΔΨm. Furthermore no change was observed in the expression level of Bcl2 pro-survival family members, while GA101 induced the pro-apoptotic proteins such as Bax and Bak and caused cleavage of the active form of caspase 9 and 3 and the proteolytic cleavage of PARP, in 5 out of 9 patients studied. Altogether, these data show that GA101 induced-cell death in B-CLL cells, unlike what has been observed in cell lines, is mediated by a caspase-dependent mechanism involving the loss of ΔΨm and the generation of ROS. Ongoing studies aim to analyze the role, the conformational changes and the cellular redistribution of Bax and Bak in response to GA101 and the modifications of other apoptosis-related proteins in CLL cells. Disclosures: No relevant conflicts of interest to declare.

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An Alternatively Spliced Form of CD79b Gene May Account for Altered B-Cell Receptor Expression in B-Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v93.7.2327.407a08_2327_2335 ◽

1999 ◽

Vol 93 (7) ◽

pp. 2327-2335 ◽

Cited By ~ 1

Author(s):

A. Alfarano ◽

S. Indraccolo ◽

P. Circosta ◽

S. Minuzzo ◽

A. Vallario ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Lines ◽

Cell Receptor ◽

B Cell Receptor ◽

Lymphocytic Leukemia ◽

Receptor Expression ◽

Rt Pcr ◽

Alternatively Spliced

Several functional anomalies of B-chronic lymphocytic leukemia (B-CLL) cells may be explained by abnormalities of the B-cell receptor (BCR), a multimeric complex formed by the sIg homodimer and the noncovalently bound heterodimer Ig/Igβ (CD79a/CD79b). Because the expression of the extracellular Ig-like domain of CD79b has been reported to be absent in the cells of most CLL cases, we have investigated the molecular mechanisms that may account for this defect. Peripheral blood lymphocytes (PBL) from 50 patients and two cell lines (MEC1, MEC2) obtained from the PBL of one of them were studied. MEC1, MEC2, and 75% of CLL cases did not express detectable levels of the extracellular Ig-like domain of CD79b, which was nevertheless present in greater than 80% CD19+ cells from normal donors. In healthy subjects the expression of CD79b was equally distributed in CD5+ and CD5− B-cell subsets. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD79b RNA from all patients and from MEC1 and MEC2 cell lines consistently yielded two fragments of different size (709 bp and 397 bp). The 709-bp band corresponds to CD79b entire transcript; the 397-bp band corresponds to an alternatively spliced form lacking exon 3 that encodes the extracellular Ig-like domain. Both fragments were also visible in normal PBL. The expression of the 397-bp fragment was increased in normal activated B cells, while no difference was seen between CD5+ and CD5− B cells. To obtain a more accurate estimate of the relative proportions of the two spliced forms, a radioactive PCR was performed in 13 normal and 22 B-CLL samples and the results analyzed using a digital imager. The mean value of the CD79b to the CD79b internally deleted ratio was 0.64 ± 0.20 SD in normal donors and 0.44 ± 0.27 SD in B-CLL (P = .01). Direct sequencing of 397-bp RT-PCR products and of genomic DNA corresponding to exon 3 from MEC1, MEC2, their parental cells, and five fresh B-CLL samples did not show any causal mutation. Single-strand conformation polymorphism analysis of exon 3 performed in 18 additional B-CLL cases showed a single abnormal shift corresponding to a TGT → TGC polymorphic change at amino acid 122. We propose a role for the alternative splicing of CD79b gene in causing the reduced expression of BCR on the surface of B-CLL cells. As normal B cells also present this variant, the mechanism of CD79b posttranscriptional regulation might reflect the activation stage of the normal B cell from which B-CLL derives.

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