cyclin-d1 and Leukemia--Lymphocytic--Chronic--B-Cell

Leukemic, non-nodal mantle cell lymphoma (MCL) is a distinct, rare, indolent variant of mantle cell lymphoma, but can relapse aggressively. It can present with lymphocytosis with chronic lymphocytic leukemia (CLL)-like morphologic and immunophenotypic features as was initially considered in the index case. However, at time of splenectomy, two years later cyclin D1 overexpression was shown and the disease was realized to be leukemic non-nodal MCL. The patient was followed for 21 years, without therapy, before he developed clinically aggressive MCL with lymphadenopathy. Lymph node biopsy showed MCL, pleomorphic variant. We review the literature and discuss the features of leukemic non-nodal MCL as well as the potential pitfalls in diagnosis. Furthermore, we are not aware of another cases reported with a 21 year interval from initial diagnosis of leukemic non-nodal MCL to aggressive MCL.

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Biopsy; Cyclin D1; Follow-Up Studies; Humans; Immunophenotyping; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphadenopathy; Lymphocytosis; Lymphoma, Mantle-Cell; Lymphoma, Non-Hodgkin; Male; Recurrence; Remission Induction; Splenectomy; Stem Cell Transplantation; Transplantation, Homologous

Chronic lymphocytic leukemia and chronic myeloid leukemia are the most common types of adult leukemia. However, it is rare for the same patient to suffer from both. Richter's transformation to diffuse large B-cell lymphoma is frequently observed in chronic lymphocytic leukemia. Purine analog therapy and the presence of trisomy 12, and CCND1 gene rearrangement have been linked to increased risk of Richter's transformation. The coexistence of chronic myeloid leukemia and diffuse large B-cell lymphoma in the same patient is extremely rare, with only nine reported cases. Here, we describe the first reported case of concurrent chronic myeloid leukemia and diffuse large B-cell lymphoma in a background of chronic lymphocytic leukemia.. A 60-year-old Saudi man known to have diabetes, hypertension, and chronic active hepatitis B was diagnosed as having Rai stage II chronic lymphocytic leukemia, with trisomy 12 and rearrangement of the CCND1 gene in December 2012. He required no therapy until January 2016 when he developed significant anemia, thrombocytopenia, and constitutional symptoms. He received six cycles of fludarabine, cyclophosphamide, and rituximab, after which he achieved complete remission. One month later, he presented with progressive leukocytosis (mostly neutrophilia) and splenomegaly. Fluorescence in situ hybridization from bone marrow aspirate was positive for translocation (9;22) and reverse transcription polymerase chain reaction detected BCR-ABL fusion gene consistent with chronic myeloid leukemia. He had no morphologic or immunophenotypic evidence of chronic lymphocytic leukemia at the time. Imatinib, a first-line tyrosine kinase inhibitor, was started. Eight months later, a screening imaging revealed new liver lesions, which were confirmed to be diffuse large B-cell lymphoma.. In chronic lymphocytic leukemia, progressive leukocytosis and splenomegaly caused by emerging chronic myeloid leukemia can be easily overlooked. It is unlikely that chronic myeloid leukemia arose as a result of clonal evolution secondary to fludarabine treatment given the very short interval after receiving fludarabine. It is also unlikely that imatinib contributed to the development of diffuse large B-cell lymphoma; rather, diffuse large B-cell lymphoma arose as a result of Richter's transformation. Fludarabine, trisomy 12, and CCND1 gene rearrangement might have increased the risk of Richter's transformation in this patient.

Topics: Antineoplastic Agents; Chromosomes, Human, Pair 12; Cyclin D1; Cyclophosphamide; Disease Progression; Gene Expression Regulation, Neoplastic; Gene Rearrangement; Hematopoietic Stem Cell Transplantation; Humans; In Situ Hybridization, Fluorescence; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukocyte Count; Lymphoma, Large B-Cell, Diffuse; Male; Middle Aged; Rituximab; Treatment Outcome; Trisomy; Vidarabine

Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma characterized by involvement of the lymph nodes, spleen, blood and bone marrow with a short remission duration to standard therapies and a median overall survival (OS) of 4-5 years.. Diagnosis is based on lymph node, bone marrow, or tissue morphology of centrocytic lymphocytes, small cell type, or blastoid variant cells. A chromosomal translocation t (11:14) is the molecular hallmark of MCL, resulting in the overexpression of cyclin D1. Cyclin D1 is detected by immunohistochemistry in 98% of cases. The absence of SOX-11 or a low Ki-67 may correlate with a more indolent form of MCL. The differential diagnosis of MCL includes small lymphocytic lymphoma, marginal zone lymphoma, and follicular lymphoma.. The MCL International Prognostic Index (MIPI) is the prognostic model most often used and incorporates ECOG performance status, age, leukocyte count, and lactic dehydrogenase. A modification of the MIPI also adds the Ki-67 proliferative index if available. The median OS for the low-risk group was not reached (5-year OS of 60%). The median OS for the intermediate risk group was 51 months and 29 months for the high risk group.. For selected indolent, low MIPI MCL patients, initial observation may be appropriate therapy. For younger patients with intermediate or high risk MIPI MCL, aggressive therapy with a cytotoxic regimen ± autologous stem cell transplantation should be considered. For older MCL patients with intermediate or high risk MIPI, combination chemotherapy with R-CHOP, R-Bendamustine, or a clinical trial should be considered. In addition, rituximab maintenance therapy may prolong the progression-free survival. At the time of relapse, agents directed at activated pathways in MCL cells such as bortezomib (NFkB inhibitor), lenalidamide (anti-angiogenesis) and Ibruitinib (Bruton's Tyrosine Kinase [BTK] inhibitor) have demonstrated excellent clinical activity in MCL patients. Autologous or allogeneic stem cell transplantation can also be considered in young patients. Clinical trials with novel agents are always a consideration for MCL patients.

Topics: Antineoplastic Combined Chemotherapy Protocols; Bone Marrow; Cyclin D1; Diagnosis, Differential; Disease Management; Hematopoietic Stem Cell Transplantation; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymph Nodes; Lymphoma, B-Cell, Marginal Zone; Lymphoma, Follicular; Lymphoma, Mantle-Cell; Risk Assessment; Spleen; Survival Analysis; Translocation, Genetic; Transplantation, Autologous

Immunophenotyping of B cell chronic lymphocytic leukaemia (B-CLL) is usually performed by flow cytometry on cell suspensions obtained from peripheral blood, bone marrow or biopsied tissue. Immunohistochemical analysis on routine sections is less commonly performed; however, this approach allows the pathologist and the researcher to appreciate the immuno-architecture of the involved tissues and to gain insight into some of the events that influence the biology of the disease. In this review the authors focus on the following issues: immuno-architecture of the proliferation centres, expression of CD23, MUM1/IRF-4 and cyclin D1, tyrosine phosphorylation and detection of the ZAP-70 kinase. Whenever possible, an attempt is made to interpret the immunohistochemical findings from a functional point of view.

Topics: Cell Proliferation; Cyclin D1; Humans; Immunophenotyping; Interferon Regulatory Factors; Leukemia, Lymphocytic, Chronic, B-Cell; Receptors, IgE; ZAP-70 Protein-Tyrosine Kinase

Cytogenetic analysis of small lymphocytes disorders is hindered by the low mitotic activity of the malignant cells. The use of fluorescence in situ hybridization (FISH) allows the detection of chromosomal amplifications, deletions, or translocations at a single-cell level in dividing and resting cells. The use of FISH in combination with other molecular techniques has defined the deletion in band 13q14 as the most common abnormality in chronic lymphocytic leukemia, followed by del (11)(q22-23), trisomy 12, del (17)(p13), and del (6)(q21). The del 13q14 is also found in 70% of mantle-cell lymphomas (MCLs) and in non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), and multiple myeloma (MM) patients. These findings point to the existence of yet unidentified tumor-suppressor gene(s) at the 13q14 locus, the loss/inactivation of which leads to B-cell neoplasia. Del (17(p13) (involving the p53 tumor-suppressor gene) and del (11)(q22-23) (involving the ataxia-telangiectasia gene [ATM]) seem to be independent prognostic factors for poor survival in chronic lymphocytic leukemia (CLL) patients. In MCL, the t(11;14) involving the bcl-1 gene is found, but data from a bcl-1 transgenic animal model suggest that hyperexpression of bcl-1 is not sufficient for lymphomatogenesis. Similar data are observed in bcl-2 transgenic animals, a finding showing that the bcl-2 hyperexpression observed in t(14;18)-positive follicular lymphoma cells is not sufficient to confer a malignant phenotype. The contribution of other chromosomal abnormalities other than bcl-1 and bcl-2 rearrangements in the pathogenesis of MCL and follicular-cell lymphomas has to be determined.

Topics: Animals; Animals, Genetically Modified; Chromosome Aberrations; Chromosome Disorders; Cyclin D1; Genes, Tumor Suppressor; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma; Tumor Suppressor Protein p53; Waldenstrom Macroglobulinemia

We have investigated the cellular origin and/or pathogenesis of follicular small cleaved cell lymphoma (FSCCL), diffuse small cleaved cell lymphoma (DSCCL) and intermediate lymphocytic lymphoma/lymphocytic lymphoma of intermediate differentiation (ILL/IDL) based on a series of immunologic and molecular genetic (bcl-1, bcl-2 and bcl-3 genes) studies. These studies have led to the conclusion that the cellular origin or pathogenesis of ILL/IDL and DSCCL is distinctly different from that of FSCCL: (1) FSCCL is a neoplastic counterpart of follicular center cells (FCC) of secondary follicles because of the presence of CD10 and bcl-2 gene rearrangement and the absence of CD5 and bcl-1 gene rearrangement; (2) DSCCL and ILL/IDL are a neoplastic counterpart of mantle zone (MZ) B lymphocytes because of the presence of CD5 and bcl-1 gene rearrangement and absence of CD10 and bcl-2 gene rearrangement; and (3) FSCCL scarcely develops into DSCCL, and the previously proposed concept that DSCCL represents a diffuse counterpart of FSCCL does not hold good. These results indicate that DSCCL and ILL/IDL are identical, derived from primary follicular cells or MZB cells of secondary follicles, and should be unified under MZB lymphocyte-derived lymphomas. They are distinguished from FCC-derived lymphomas in morphologic, immunologic, cytogenetic and molecular genetic features. Bcl-1 and bcl-2 genes may be associated with the pathogenesis of FCC-derived lymphoma and MZB lymphocyte-derived lymphoma, respectively.

Topics: Antigens, CD; Cyclin D1; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Follicular; Lymphoma, Non-Hodgkin; Phenotype; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2

In the work it has been decided to evaluate the occurrence of locus bcl-1 rearrangement in type B chronic lymphatic leukemia and that of gene bcl-2 in non-Hodgkin's lymphoma with diffuse morphology, as well as in reactive lymph nodes. The study material comprised DNA isolated from fragments of lymph nodes sent for routine diagnostic examinations at the Institute of Pathology--Pomeranian Medical Academy. Southern's method was used to examine DNA having been cut with restrictive enzymes, estimating the distribution of gene bcl-2 and locus bcl-1. Resorting to Polymerase Chain Reaction (PCR) translocation t (14;18) was assessed by means of short nucleotides hybridizing with 14 and 18 chromosome sequences restricting this translocation. The amplification product was subsequently studied by Southern's method with probe bcl-2. In 1 out of 18 examined cases of type B chronic lymphocyte leukemia it was disclosed that locus bcl-1 had been rearranged. In 45 cases of non-Hodgkin's lymphoma with diffuse morphology the gen bcl-2 was found to display germline arrangement. Germline position of gen bcl-2 was also revealed in 60 cases of reactive lymph nodes.

Topics: Blotting, Southern; Chromosomes, Human, Pair 14; Chromosomes, Human, Pair 18; Cyclin D1; Digoxigenin; DNA, Neoplasm; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Non-Hodgkin; Polymerase Chain Reaction; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Translocation, Genetic

Topics: Aged; Chromosomes, Human, Pair 14; Chromosomes, Human, Pair 6; Cyclin D1; Cyclin D3; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Mantle-Cell; Male; Oncogene Proteins, Fusion; Translocation, Genetic

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) occasionally undergoes Richter transformation, mostly to diffuse large B-cell lymphoma, but its evolution to other types of B-cell lymphoma is rare. We report a CLL evolved to mantle cell lymphoma by acquiring t(11;14)(q13;q32); CCND1-IGH.. A Retrospective review of clinical and laboratory data.. A 39-year-old male patient was diagnosed with CLL/SLL, and was initially followed without specific treatment, but subsequently received chlorambucil/fludarabine/rituximab due to exacerbated lymphocytosis. While his CLL/SLL waned and waxed, the immunophenotype and genotype of neoplastic B-cells remained unchanged, without cyclin D1 expression and CCND1-IGH fusion. Eleven years after the diagnosis, the patient's disease showed evidence of progression. Bone marrow examination demonstrated "CLL" with the morphology and immunophenotype similar to those seen in the previous biopsies. Unexpectedly, the neoplastic B-cells demonstrated cyclin D1 expression and harbored t(11;14)(q13;q32); CCND1-IGH, suggesting a clonal evolution to mantle cell lymphoma. He subsequently received cytoreductive chemotherapy followed by allogenic bone marrow transplant and remained in remission since then.. The retention of immunophenotype suggests a clonal relationship between CLL/SLL and mantle cell lymphoma. While the acquisition of t(11;14)(q13;q32); CCND1-IGH likely alters the disease course, the pathogenesis of this illegitimate translocation in CLL remains to be studied.

Topics: Adult; Cyclin D1; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Large B-Cell, Diffuse; Lymphoma, Mantle-Cell; Male; Oncogene Proteins, Fusion; Translocation, Genetic

Multiple myeloma is a genetically heterogeneous cancer of the bone marrow plasma cells (PC). Distinct myeloma transcriptome profiles are primarily driven by myeloma initiating events (MIE) and converge into a mutually exclusive overexpression of the CCND1 and CCND2 oncogenes. Here, with reference to their normal counterparts, we find that myeloma PC enhanced chromatin accessibility combined with paired transcriptome profiling can classify MIE-defined genetic subgroups. Across and within different MM genetic subgroups, we ascribe regulation of genes and pathways critical for myeloma biology to unique or shared, developmentally activated or de novo formed candidate enhancers. Such enhancers co-opt recruitment of existing transcription factors, which although not transcriptionally deregulated per se, organise aberrant gene regulatory networks that help identify myeloma cell dependencies with prognostic impact. Finally, we identify and validate the critical super-enhancer that regulates ectopic expression of CCND2 in a subset of patients with MM and in chronic lymphocytic leukemia.

Topics: Bone Marrow Cells; Carcinogenesis; Case-Control Studies; Cell Line, Tumor; Chromatin; Cyclin D1; Cyclin D2; Enhancer Elements, Genetic; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Histone-Lysine N-Methyltransferase; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Multiple Myeloma; Plasma Cells; Proto-Oncogene Proteins c-maf; Repressor Proteins; Survival Analysis; Transcriptome

The clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) identifies and tracks unique disease-associated immunoglobulin (Ig) sequences by next-generation sequencing of IgH, IgK, and IgL rearrangements and IgH-BCL1/2 translocations in malignant B cells. Here, we describe studies to validate the analytical performance of the assay using patient samples and cell lines.. Sensitivity and specificity were established by defining the limit of detection (LoD), limit of quantitation (LoQ) and limit of blank (LoB) in genomic DNA (gDNA) from 66 patients with multiple myeloma (MM), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL), and three cell lines. Healthy donor gDNA was used as a diluent to contrive samples with specific DNA masses and malignant-cell frequencies. Precision was validated using a range of samples contrived from patient gDNA, healthy donor gDNA, and 9 cell lines to generate measurable residual disease (MRD) frequencies spanning clinically relevant thresholds. Linearity was determined using samples contrived from cell line gDNA spiked into healthy gDNA to generate 11 MRD frequencies for each DNA input, then confirmed using clinical samples. Quantitation accuracy was assessed by (1) comparing clonoSEQ and multiparametric flow cytometry (mpFC) measurements of ALL and MM cell lines diluted in healthy mononuclear cells, and (2) analyzing precision study data for bias between clonoSEQ MRD results in diluted gDNA and those expected from mpFC based on original, undiluted samples. Repeatability of nucleotide base calls was assessed via the assay's ability to recover malignant clonotype sequences across several replicates, process features, and MRD levels.. LoD and LoQ were estimated at 1.903 cells and 2.390 malignant cells, respectively. LoB was zero in healthy donor gDNA. Precision ranged from 18% CV (coefficient of variation) at higher DNA inputs to 68% CV near the LoD. Variance component analysis showed MRD results were robust, with expected laboratory process variations contributing ≤3% CV. Linearity and accuracy were demonstrated for each disease across orders of magnitude of clonal frequencies. Nucleotide sequence error rates were extremely low.. These studies validate the analytical performance of the clonoSEQ Assay and demonstrate its potential as a highly sensitive diagnostic tool for selected lymphoid malignancies.

Topics: Bone Marrow; Cyclin D1; Gene Rearrangement; High-Throughput Nucleotide Sequencing; Humans; Immunoglobulin Heavy Chains; Immunoglobulin lambda-Chains; Immunoglobulins; Leukemia, Lymphocytic, Chronic, B-Cell; Limit of Detection; Multiple Myeloma; Neoplasm, Residual; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-bcl-2; Reagent Kits, Diagnostic; Translocation, Genetic

Topics: Aged; Bone Marrow; Cyclin D1; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoid Enhancer-Binding Factor 1; Male; Melanoma; Neoplasms, Second Primary

Topics: Cyclin D1; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Translocation, Genetic

Cyclin D1, generally considered to be absent in chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL), has been reported in the proliferation centres (PCs) of recent CLL/SLL cases. Cyclin D1 immunostaining in CLL/SLL may lead to diagnostic confusion. The objective of this study was to identify the types of stained cells and the impact on diagnosis.. Cyclin D1 expression was assessed by immunostaining samples from 46 cases of CLL/SLL. CD68 and double immunostaining with CD20/CyclinD1, CD68/CyclinD1, and CD163/CyclinD1 were then performed in cases of CLL/SLL positive for cyclinD1 in the PCs.. Dim-positive cyclin D1 staining in randomly scattered cells in the CLL/SLLs were observed in 38/46 cases (82.6%). In five (10.9%) cases, more than 50 cyclin D1-positive cells per high-power field were detected within the PCs in CLL/SLL with weak to moderate intensity. Double immunochemical staining in these cases showed that cyclin D1 in these positive cells was mostly co-expressed with CD68 and CD163 and the cells were negative for CD20.. The cyclin D1-positive CLL/SLL cells in this study were mostly histiocytes. The expression of cyclin D1 by histiocytes may mimic cyclin D1+ CLL/SLL; thus, the recognition of cyclin D1 expression by non-lymphoid cells in lymphoma is important.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Cell Proliferation; Cyclin D1; Diagnosis, Differential; Female; Histiocytes; Humans; Immunohistochemistry; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Mantle-Cell; Male; Middle Aged

The recent publication reviewing the updated WHO classification commented on the presence of cyclin D1-positive cells in the proliferation centres (PC) of small lymphocytic lymphoma/chronic lymphocytic leukaemia (SLL/CLL). The figure quoted was 30%, which appeared higher than our experience. To assess cyclin D1 expression in PC of SLL/CLL cases, we performed a review of SLL/CLL cases diagnosed at the Royal Marsden Hospital between 1996 and 2009. Of 105 SLL/CLL cases, 16.2% showed expression of cyclin D1 in PC with none carrying the translocation t(11;14)(q13;q32). Our study and a review of the published literature suggest that this phenomenon occurs with a significantly lower prevalence than that described in the recent review of the updated WHO classification. We confirm that cyclin D1 expression is confined to PC with the typical small lymphocytes being negative. This finding is apparently unrelated to the translocation involving

Topics: Adult; Aged; Aged, 80 and over; Cyclin D1; Female; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged

Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries, with incidence in Chinese populations also increasing. CLL involves an accumulation of abnormal B cells which result in dysregulation of cell proliferation and apoptosis rates. The calcyclin-binding protein/Siah-1-interacting protein (CacyBP/SIP) plays a pivotal role in tumorigenicity and cell apoptosis. Here, we investigated the function of CacyBP/SIP in CLL cell proliferation and apoptosis.. CacyBP/SIP expression levels were measured in peripheral blood mononuclear cells from 23 Chinese CLL patients and three healthy donors by western blotting. Correlation analysis was performed to assess associations between CacyBP/SIP expression and clinical stage, chromosome abnormalities and zeta-chain-associated protein kinase 70 (ZAP-70) expression. We silenced CacyBP/SIP expression in MEC-1 cells using a lentivirus system and analyzed cell vitality, cell cycle and tumorigenicity. Apoptosis was also analyzed following the upregulation of CacyBP/SIP expression in MEC-1 cells.. Downregulation of CacyBP/SIP expression in CLL patients was negatively correlated with CLL clinical stage, but not with patient sex, age, del(13q14) or del(17q-) presence, or ZAP-70 expression. CacyBP/SIP silencing significantly enhanced cell proliferation and tumorigenicity. CacyBP/SIP silencing promoted accumulation of cells in S phase by upregulation of β-catenin, cyclin D1 and cyclin E, and downregulation of p21. Moreover, CacyBP/SIP overexpression facilitated CLL apoptosis through the activation of pro-caspase-3.. CacyBP/SIP is a useful indicator of CLL disease processes and plays an important role in sustaining the balance of cell proliferation and apoptosis.

Topics: Animals; Apoptosis; Asian People; beta Catenin; Blotting, Western; Calcium-Binding Proteins; Caspase 3; Cell Proliferation; Cyclin D1; Cyclin E; Down-Regulation; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Staging; S Phase; Signal Transduction; Up-Regulation

T(12;14)(p13;q32) is a rare recurrent chromosomal translocation, which has only been identified in a small subgroup of mantle cell lymphoma (MCL) without typical t(11;14)(q13;q32). This rearrangement causes aberrant over-expression of cyclin D2 (CCND2), which disrupts the normal cell cycle. Here we report a subtle case of MCL with t(12;14)(p13;q32) that was initially misdiagnosed as ultra-high risk chronic lymphocytic leukemia (CLL). A 60-year-old male patient presented with obvious leukocytosis and progressive weakness. Morphology of peripheral blood and immunophenotyping by flow cytometry pointed to a diagnosis of chronic lymphocytic leukemia. Fluorescence in situ hybridization (FISH) using IGH-CCND1 probe was negative for CCND1 abnormality, but demonstrated IGH breakapart signals. The initial diagnosis of CLL was established and the patient was treated with six courses of immunochemotherpy with fludarabine, cyclophosphamide and rituximab (FCR). Complete remission (CR) was achieved at the end of treatment, but disease relapsed quickly. The patient was transferred to our hospital, flow cytometry using additional markers showed that the clonal cells were CD200+(dim), CD148+(strong), and chromosome analysis revealed a complex karyotype, 47, XY, t(12;14)(p13;q32), +12, del(9p21), which indicated over-expression of CCND2, and immunostaining showed strong positivity of SOX11 further confirming the characteristics of CCND1-negtive MCL. The final diagnosis was revised to rare subtype of MCL with CCND2 translocation and intensive regimens were employed. This confusable MCL case illustrates the importance of cytogenetic analysis and clinicopathologic diagnosis of this rare category of MCL.

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Bone Marrow Examination; Chromosomes, Human, Pair 12; Chromosomes, Human, Pair 14; Cyclin D1; Diagnosis, Differential; Diagnostic Errors; Disease Progression; Fatal Outcome; Genes, Immunoglobulin Heavy Chain; Humans; Immunohistochemistry; Immunophenotyping; In Situ Hybridization, Fluorescence; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Mantle-Cell; Male; Middle Aged; Predictive Value of Tests; Risk Factors; Time Factors; Translocation, Genetic; Treatment Outcome

The central role of the microRNA (miR) 15a/16-1 cluster in B-cell oncogenesis has been extensively demonstrated, with over two-thirds of B-cell chronic lymphocytic leukemia characterized by the deletion of the miR-15a/16-1 locus at 13q14. Despite the well-established understanding of the molecular mechanisms occurring during miR-15a/16-1 dysregulation, the oncogenic role of other miR-15/16 family members, such as the miR-15b/16-2 cluster (3q25), is still far from being elucidated. Whereas miR-15a is highly similar to miR-15b, miR-16-1 is identical to miR-16-2; thus, it could be speculated that both clusters control a similar set of target genes and may have overlapping functions. However, the biological role of miR-15b/16-2 is still controversial. We generated miR-15b/16-2 knockout mice to better understand the cluster's role in vivo. These mice developed B-cell malignancy by age 15-18 mo with a penetrance of 60%. At this stage, mice showed significantly enlarged spleens with abnormal B cell-derived white pulp enlargement. Flow cytometric analysis demonstrated an expanded CD19+ CD5+ population in the spleen of 40% knockout mice, a characteristic of the chronic lymphocytic leukemia-associated phenotype found in humans. Of note, miR-15b/16-2 modulates the CCND2 (Cyclin D2), CCND1 (Cyclin D1), and IGF1R (insulin-like growth factor 1 receptor) genes involved in proliferation and antiapoptotic pathways in mouse B cells. These results are the first, to our knowledge, to suggest an important role of miR-15b/16-2 loss in the pathogenesis of B-cell chronic lymphocytic leukemia.

Topics: Animals; Cyclin D1; Cyclin D2; Gene Deletion; Gene Expression Profiling; Gene Expression Regulation, Leukemic; HEK293 Cells; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Mice; Mice, Knockout; MicroRNAs; Receptor, IGF Type 1

High dose methylprednisolone (HDMP) has been an effective salvage therapy for patients with relapsed chronic lymphocytic leukemia (CLL), while little is known about the exact mechanisms implicated in glucocorticoid-induced cell death. To explore the mechanism of glucocorticoid-induced cell death, we investigated the effect of HDMP on canonical Wnt signaling which emerged as a key pathway implicated in the pathogenesis of CLL. In this study, the human CLL cell line MEC-1 was incubated with various concentrations of methylprednisolone. Cell proliferation activity was detected by CCK8 assay, the apoptotic effect was evaluated by TUNEL assay. Western blot was used to detect active-caspase 3, and the key proteins in Wnt signaling pathway (LEF-1, β-catenin). RT-PCR was performed to assess the mRNA levels of β-catenin, LEF-1, c-myc and cyclin D1. We observed that high concentration of methylprednisolone could suppress the proliferation activity of MEC-1 cells, promote the relative expression of active-caspase 3, and induce apoptotic cell death. Furthermore, methylprednisolone could inhibit LEF-1 protein expression, consequently down-regulate mRNA levels of c-myc and cyclin D1, but could not affect the transcription level of β-catenin and LEF-1 mRNA. The results of this study indicate that methylprednisolone can suppress Wnt signaling pathway by down-regulating LEF-1 protein expression, indicating a novel mechanism for HDMP therapy in CLL.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoid Enhancer-Binding Factor 1; Methylprednisolone; Proto-Oncogene Proteins c-myc; RNA, Messenger; Time Factors; Up-Regulation; Wnt Signaling Pathway

Clinical trials of heat shock protein 90 (Hsp90) inhibitors have been limited by high toxicity. We previously showed that the Hsp90 inhibitor, SNX-7081, synergizes with and restores sensitivity to fludarabine nucleoside (2-FaraA) in human chronic lymphocytic leukemia (CLL) cells with lesions in the p53 pathway (Best OG, et al., Leukemia Lymphoma 53:1367-75, 2012). Here, we used label-free quantitative shotgun proteomics and comprehensive bioinformatic analysis to determine the mechanism of this synergy. We propose that 2-FaraA-induced DNA damage is compounded by SNX-7081-mediated inhibition of DNA repair, resulting in enhanced induction of apoptosis. DNA damage responses are impaired in part due to reductions in checkpoint regulators BRCA1 and cyclin D1, and cell death is triggered following reductions of MYC and nucleolin and an accumulation of apoptosis-inducing NFkB2 p100 subunit. Loss of nucleolin can activate Fas-mediated apoptosis, leading to the increase of pro-apoptotic proteins (BID, fas-associated factor-2) and subsequent apoptosis of p53-negative, 2-FaraA refractory CLL cells. A significant induction of DNA damage, indicated by increases in DNA damage marker γH2AX, was observed following the dual drug treatment of additional cell lines, indicating that a similar mechanism may operate in other p53-mutated human B-lymphoid cancers. These results provide valuable insight into the synergistic mechanism between SNX-7081 and 2-FaraA that may provide an alternative treatment for CLL patients with p53 mutations, for whom therapeutic options are currently limited. Moreover, this drug combination reduces the effective dose of the Hsp90 inhibitor and may therefore alleviate any toxicity encountered.

Topics: Antineoplastic Agents; Apoptosis; Benzamides; Blotting, Western; BRCA1 Protein; Cell Line, Tumor; Chromatography, Liquid; Cyclin D1; DNA Damage; DNA Repair; Drug Synergism; HSP90 Heat-Shock Proteins; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Mutation; NF-kappa B p52 Subunit; Nucleolin; Phosphoproteins; Protein Interaction Maps; Proteomics; Proto-Oncogene Proteins c-myc; RNA-Binding Proteins; Signal Transduction; Tandem Mass Spectrometry; Tumor Suppressor Protein p53; Vidarabine

BMI1, a Polycomb-group gene located at 10p12.2, is implicated in the pathogenesis of a variety of tumors. However, the genetic molecular mechanisms underlying its aberrant expression in cancer cells remain largely unknown. In this study, we show that BMI1 is recurrently targeted by chromosomal aberrations in B-cell leukemia/lymphoma. We identified a novel t(10;14)(p12;q32)/IGH-BMI1 rearrangement and its IGL variant in six cases of chronic lymphocytic leukemia (CLL) and found that these aberrations were consistently acquired at time of disease progression and high grade transformation of leukemia (Richter syndrome). The IG-BMI1 translocations were not associated with any particular molecular subtype of CLL and the leukemias were negative for common mutations of NOTCH1 and TP53, known to increase a risk of progression and transformation in CLL. In addition, using FISH and SNP array analysis, we identified a wide range of BMI1-involving 10p12 lesions in 17 cases of mantle cell lymphoma (MCL). These aberrations included various balanced and unbalanced structural abnormalities and very frequently but not exclusively, were associated with gain of the BMI1 locus and loss of the 10p terminal sequences. These findings point to genomic instability at the 10p region in MCL which likely promotes rearrangements and deregulation of BMI1. Our findings are in line with previously published observations correlating overexpression of BMI1 with tumor progression and chemoresistance. In summary, our study provides new insights into genetic molecular mechanisms underlying aberrant expression of BMI1 in lymphoma and documents its contribution in the pathogenesis of Richter syndrome and MCL.

Topics: Aged; Aged, 80 and over; Allelic Imbalance; Chromosome Aberrations; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; In Situ Hybridization, Fluorescence; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Male; Middle Aged; Polycomb Repressive Complex 1; Polymorphism, Single Nucleotide; Translocation, Genetic

SOX11 is mainly correlated with embryo neurogenesis and remodeling of tissues. D cyclins (cyclin D1, cyclin D2, and cyclin D3) work in cell transformation. We assessed the expression of SOX11, cyclin D1, cyclin D2, and cyclin D3 mRNA in 152 patients with B-cell lymphocytic proliferative diseases (B-LPD) using qRT-PCR and we detected SOX11 protein using immunohistochemistry in 15 B-LPD patients, to clarify the clinical significance of the four genes in B-LPD. Data showed the transcriptional levels of SOX11 and cyclin D1 were higher for the mantle cell lymphoma (MCL) samples compared with chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), hairy cell leukemia (HCL), splenic marginal zone lymphoma (SMZL), and healthy collators. The expression levels of cyclin D1 and cyclin D2 were both higher in DLBCL than in SMZL. The expression levels of the four genes were highly related to each other. Three of 4 MCL patients showed nuclear staining for SOX11, while other 11 B-LPD examples were negative. Furthermore, we also found the ZAP70-positive CLL patients had higher SOX11 expression levels than ZAP70-negative CLL patients. It was revealed that MCL patients have higher expression levels of SOX11 and cyclin D1 mRNA, specially expressed nuclear SOX11 protein.

Topics: Adult; Aged; Aged, 80 and over; Case-Control Studies; Cyclin D1; Cyclin D2; Cyclin D3; Female; Humans; Immunoenzyme Techniques; Leukemia, Hairy Cell; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Large B-Cell, Diffuse; Lymphoma, Mantle-Cell; Male; Middle Aged; Prognosis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; SOXC Transcription Factors

Cyclin D1 expression, usually absent in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), has been described in the proliferation centers (PC) of some CLL/SLL. The prevalence of this finding is uncertain, as is the explanation for its occurrence and whether these cases have any other unique features. Cyclin D1 immunohistochemical staining was therefore investigated in 57 extramedullary CLL/SLL biopsies. In 6 cases, cyclin D1 immunofluorescence followed by CCND1 fluorescence in situ hybridization (FISH) and PC targeted analysis was performed using a Bioview Duet system. Excluding the prospectively selected cases that had the targeted FISH studies, cyclin D1+ PC were identified in 20% of cases. The cyclin D1+ CLL did not appear pathologically or phenotypically distinctive, though 46% had an interfollicular growth pattern. The cyclin D1+ PCs were SOX11- and lacked CCND1 translocations and gains in 5 of 5 informative cases. The recognition of cyclin D1 expression in PC of a significant minority of CLL/SLL can be a diagnostic aid and should not lead to the diagnosis of focal mantle cell lymphoma.

Topics: Aged; Aged, 80 and over; Cyclin D1; Female; Germinal Center; Humans; In Situ Hybridization, Fluorescence; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; SOXC Transcription Factors; Translocation, Genetic

Interphase fluorescence in situ hybridization (FISH) can identify submicroscopic deletions adjacent to the breakpoints of rearrangements undetected by conventional cytogenetics. In this study, the characteristics and frequency of the IgH deletion identified by interphase FISH were investigated in patients with multiple myeloma (MM) and chronic lymphocytic leukemia (CLL).. The study group included 29 patients with MM and eight patients with CLL. Interphase FISH was performed with the IgH dual color, break-apart rearrangement probe and the IgH/CCND1 dual color, dual fusion translocation probe.. The IgH deletion was found in 14% (4/29) of patients with MM and 13% (1/8) of the patients with CLL. Four patients had deletions of the whole or variable region of IgH on the native chromosome 14, whereas one patient had a deletion of the IgH variable region on a der(11)t(11;14). In two patients, the IgH break-apart FISH showed both patterns with and without IgH deletions. In cases showing the same pattern by IgH break-apart FISH, the IgH/CCND1 FISH showed different patterns, and vice versa.. A variety of patterns of the IgH deletion were identified by interphase FISH using IgH break-apart and IgH/CCND1 probes in patients with MM and CLL. The results of this study suggest that the integrated information obtained with IgH break-apart and IgH/CCND1 FISH was needed to interpret FISH results unambiguously.

Topics: Aged; Chromosomes, Human, Pair 14; Cyclin D1; Female; Gene Deletion; Humans; Immunoglobulin Heavy Chains; In Situ Hybridization, Fluorescence; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Multiple Myeloma; Translocation, Genetic

Mantle cell lymphoma (MCL) is recognized as a well-defined B cell neoplasm characterized by overexpression of cyclin D1 (CCND1), with "classical" and "aggressive" variant subtypes. A small-cell variant of MCL (small-MCL), resembling small lymphocytic lymphoma/chronic lymphocytic lymphoma (CLL/SLL), has been added to the World Health Organization classification. However, to the best of our knowledge, there have been no studies focusing on this neoplasm. In the present study, we analyzed 15 cases of CCND1-positive small-MCL, including immunohistochemical analysis of Ki-67 and CCND1 expression, and compared our findings with those of 151 cases of classical MCL. Morphologically, most small-MCL showed a diffuse growth pattern (76.9%), whereas others featured a very thin mantle zone pattern resembling a reactive follicle (23.1%). Bone marrow involvement and splenomegaly occurred significantly more frequently in small-MCL than in classical MCL (P < 0.05). Ki-67 expression in small-MCL was lower than in classical MCL (mean [± 2 SD] 12.5 ± 17.3% and 25.2 ± 25.5%, respectively; P < 0.001), but there was no significant difference in CCND1 expression (P = 0.2445). The 5-year survival rate in small-MCL was 83.3%. Although there was no significant difference in outcome between small-MCL and classical MCL (P = 0.287), only one small-MCL patient died of the disease. Thus, small-MCL constitutes a specific subset of indolent lymphoma with distinguishing features, possibly making a major contribution to the accuracy of therapeutic decisions. In addition, clinicians should be aware of the possible presence of small-MCL to avoid making a misdiagnosis of follicular hyperplasia or CLL/SLL.

Topics: Aged; Bone Marrow Neoplasms; Cyclin D1; Female; Humans; Ki-67 Antigen; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Mantle-Cell; Male; Middle Aged; Splenomegaly

Topics: Antigens, CD20; CD5 Antigens; Colonic Neoplasms; Cyclin D1; Diagnosis, Differential; Female; Humans; Ileal Diseases; Ileocecal Valve; Intestinal Neoplasms; Intestinal Polyps; Intussusception; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Mantle-Cell; Middle Aged

Cyclin D1, an important component of cell cycle machinery and a protein with known oncogenic potential, is downregulated in normal mature B lymphocytes. Its expression detected in a number of malignancies, including B-cell lymphomas, may be important for oncogenesis.. In our work, we determined the level of cyclin D1 expression in various B-cell lymphomas (i.e., mantle cell lymphoma, B-cell chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and marginal zone lymphoma) and compared it with normal B cells. For cyclin D1 level evaluation, the real-time quantitative polymerase chain reaction data was normalized. We tested five reference genes for stability on our sample set and using the three most stable ones (YWHAZ, ubiquitin c, and HPRT) obtained rather small intra-group variance for cyclin D1 expression in most lymphomas. This allowed their statistically significant ranking according to cyclin D1 expression level.. Median values of normalized cyclin D1 expression determined by real-time quantitative polymerase chain reaction were 1.32 for mantle cell lymphoma, 0.02 for B-cell chronic lymphocytic leukemia, 0.009 for diffuse large B-cell lymphoma, 0.004 for marginal zone lymphoma, 0.002 for follicular lymphoma compared with 0.0003 for reactive lymphoid tissue, and 0.00004 for sorted B cells of healthy donors.. Our data demonstrate that mantle cell lymphoma, a lymphoma with t(11;14)(q13;q32) translocation, has the level of cyclin D1 increased by four orders of magnitude, while other B-cell lymphomas without t(11;14)(q13;q32) translocation still have the level of cyclin D1 significantly elevated above that of normal lymphocytes (2 orders for B-cell chronic lymphocytic leukemia and an order for other lymphomas) and suggests more than one method of its upregulation in malignant B cells.

Topics: Aged; B-Lymphocytes; Cyclin D1; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Hyperplasia; Immunohistochemistry; Leukemia, Lymphocytic, Chronic, B-Cell; Leukocytes, Mononuclear; Lymph Nodes; Lymphoma, B-Cell; Lymphoma, B-Cell, Marginal Zone; Lymphoma, Follicular; Lymphoma, Large B-Cell, Diffuse; Lymphoma, Mantle-Cell; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; Spleen

The incidence of chronic lymphocytic leukemia is low in the Japanese population compared with populations in western countries, suggesting a role for genetic factors in the occurrence of this disease. We have previously shown that chronic lymphocytic leukemia in Japan rarely expresses the immunoglobulin heavy chain variable region (IGHV) 1-69 gene (1 out of 43 patients, 2.3%), which is a gene most commonly expressed in chronic lymphocytic leukemia cases from western countries. In the current study, we extended the previous study by examining immunoglobulin heavy chain and light chain gene expression in 80 Japanese patients with chronic lymphocytic leukemia and in 52 Japanese patients with other leukemic chronic lymphoproliferative disorders. IGHV1-69 gene expression was again quite low in our cohort, found in only two patients: one with chronic lymphocytic leukemia and the other with splenic marginal zone lymphoma. The IGHV4-34 gene was most frequently expressed in chronic lymphocytic leukemia (27.5%), whereas it was rarely found in leukemic chronic lymphoproliferative disorders (7.7%, P = 0.005). There was also a significant difference in the expression of IGLV3-21 between chronic lymphocytic leukemia and leukemic chronic lymphoproliferative disorders (29.4 vs 4.8%, P = 0.018). The IGLV3-21 gene in the majority of chronic lymphocytic leukemia cases was associated with homologous complementarity determining region 3 sequences. Recent studies identified subsets of cases expressing almost identical B-cell receptors. We found that two patients with chronic lymphocytic leukemia and the patient with splenic marginal zone lymphoma expressed IGHV4-39/IGKV1-39 and IGHV1-69/IGKV3-20, respectively, which belong to these subsets.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD19; CD5 Antigens; Cohort Studies; Cyclin D1; Humans; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Immunoglobulin Variable Region; Japan; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoproliferative Disorders; Middle Aged; Mutation; Receptors, IgE

Topics: Animals; Antineoplastic Agents; Catechin; Clinical Trials as Topic; Cyclin D1; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; National Cancer Institute (U.S.); Neoplasms; Signal Transduction; United States; United States Food and Drug Administration

Demonstration of cyclin D1 expression by immunohistochemistry in a CD5-positive small B-cell proliferation is extremely helpful in the diagnosis of mantle cell lymphoma in tissue samples, including bone marrow trephine biopsies (BMTB) and in differentiating them from chronic lymphocytic leukaemia (CLL). Following the identification of cyclin D1 expression in one case of CLL on BMTB, 64 additional cases, which included 25 lymph nodes, one tonsillar lesion, one skin lesion and 37 BMTBs were systematically reviewed for presence of cyclin D1 overexpression. Overall, in seven of 65 samples (approximately 10%) of CLL, a minority of the leukaemic cells in the proliferation centres expressed cyclin D1. Cytogenetic analysis had been performed in three of seven cases and there was no evidence of translocation involving CCND1 locus. Our findings suggest that a small subset of CLL overexpresses cyclin D1 in amounts that can be demonstrated by immunohistochemistry. Our observation has impact on the diagnosis of small B-cell lymphomas in BMTB and other tissue samples.

Topics: Biopsy; Bone Marrow; CD5 Antigens; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Leukemic; Humans; Immunohistochemistry; Leukemia, Lymphocytic, Chronic, B-Cell; Lymph Nodes; Middle Aged; Phosphorylation

Alterations in the human 13q14 genomic region containing microRNAs mir-15a and mir-16-1 are present in most human chronic lymphocytic leukemia (CLL). We have previously found the development of CLL in the New Zealand Black murine model to be associated with a point mutation in the primary mir-15a/16-1 region, which correlated with a decrease in mature miR-16 and miR-15a levels. In this study, addition of exogenous miR-15a and miR-16 led to an accumulation of cells in G(1) in non-New Zealand Black B cell and New Zealand Black-derived malignant B-1 cell lines. However, the New Zealand Black line had significantly greater G(1) accumulation, suggesting a restoration of cell cycle control upon exogenous miR-15a/16 addition. Our experiments showed a reduction in protein levels of cyclin D1, a miR-15a/16 target and cell cycle regulator of G(1)/S transition, in the New Zealand Black cell line following miR-15a/16 addition. These microRNAs were shown to directly target the cyclin D1 3' untranslated region using a green fluorescent protein lentiviral expression system. miR-16 was also shown to augment apoptosis induction by nutlin, a mouse double minute 2 (MDM2) antagonist, and genistein, a tyrosine kinase inhibitor, when added to a B-1 cell line derived from multiple in vivo passages of malignant B-1 cells from New Zealand Black mice with CLL. miR-16 synergized with nutlin and genistein to induce apoptosis. Our data support a role for the mir-15a/16-1 cluster in cell cycle regulation and suggest that these mature microRNAs in both the New Zealand Black model and human CLL may be targets for therapeutic efficacy in this disease.

Topics: Animals; Antineoplastic Agents; Apoptosis; Base Sequence; Cyclin D1; Disease Models, Animal; DNA Primers; Drug Screening Assays, Antitumor; Genistein; Imidazoles; Leukemia, Lymphocytic, Chronic, B-Cell; Mice; Mice, Inbred C57BL; MicroRNAs; Piperazines; RNA, Messenger

Aberrant activation of Wnt/beta-catenin signaling promotes the development of several cancers. It has been demonstrated that the Wnt signaling pathway is activated in chronic lymphocytic leukemia (CLL) cells, and that uncontrolled Wnt/beta-catenin signaling may contribute to the defect in apoptosis that characterizes this malignancy. Thus, the Wnt signaling pathway is an attractive candidate for developing targeted therapies for CLL.. The diuretic agent ethacrynic acid (EA) was identified as a Wnt inhibitor using a cell-based Wnt reporter assay. In vitro assays further confirmed the inhibitory effect of EA on Wnt/beta-catenin signaling. Cell viability assays showed that EA selectively induced cell death in primary CLL cells. Exposure of CLL cells to EA decreased the expression of Wnt/beta-catenin target genes, including LEF-1, cyclin D1 and fibronectin. Immune co-precipitation experiments demonstrated that EA could directly bind to LEF-1 protein and destabilize the LEF-1/beta-catenin complex. N-acetyl-L-cysteine (NAC), which can react with the alpha, beta-unsaturated ketone in EA, but not other anti-oxidants, prevented the drug's inhibition of Wnt/beta-catenin activation and its ability to induce apoptosis in CLL cells.. Our studies indicate that EA selectively suppresses CLL survival due to inhibition of Wnt/beta-catenin signaling. Antagonizing Wnt signaling in CLL with EA or related drugs may represent an effective treatment of this disease.

Topics: Acetylcysteine; beta Catenin; Cell Line, Tumor; Cell Survival; Cyclin D1; Drug Screening Assays, Antitumor; Ethacrynic Acid; Fibronectins; Frizzled Receptors; Gene Expression Regulation, Leukemic; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoid Enhancer-Binding Factor 1; Protein Binding; Receptors, G-Protein-Coupled; Signal Transduction; Wnt Proteins

Wnt signalling has recently been implicated in the pathogenesis of cancer.. This study investigated the activity of Wnt signalling in peripheral blood chronic lymphocytic leukaemia (CLL) lymphocytes, and the methylation status of seven soluble Wnt antagonist genes, including WIF1, DKK3, APC, SFRP1, SFRP2, SFRP4 and SFRP5, by using methylation-specific PCR in the peripheral blood CLL lymphocytes and bone marrow samples of patients with CLL at diagnosis.. In the peripheral blood CLL lymphocytes, constitutive activation of Wnt signalling was detected, associated with hypermethylation of the soluble Wnt inhibitor genes. In the diagnostic CLL marrow samples, methylation of the seven genes was detected in up to 36.4% of samples. Moreover, 23 (52.3%) patients had methylation of at least one of the seven genes, of whom 14 (60.8%) had methylation of two or more Wnt inhibitor genes. Apart from an association of advanced age with DKK3 methylation, there was no association of gene hypermethylation with either clinical characteristics (including age, gender, lymphocyte count at diagnosis, Rai stage and poor-risk karyotype) or survival.. Wnt signalling is constitutively activated in CLL B lymphocytes in association with methylation of multiple soluble Wnt antagonist genes. Methylation of these soluble Wnt antagonist genes, occasionally multiple genes, in primary CLL marrow samples suggests an important role in CLL pathogenesis. Moreover, this study underscored the importance of studying methylation of a panel of, but not individual, genes regulating a cellular pathway.

Topics: Adult; Aged; Aged, 80 and over; B-Lymphocytes; Base Sequence; beta Catenin; Blotting, Western; Bone Marrow; Cyclin D1; DNA Methylation; DNA, Neoplasm; Epigenesis, Genetic; Female; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Leukocytes, Mononuclear; Male; Middle Aged; Molecular Sequence Data; Neoplasm Proteins; Polymerase Chain Reaction; Signal Transduction; Wnt Proteins

We analyzed protein expression of cyclin D1, cyclin D2, and cyclin D3 using high-resolution enzymatic amplification staining and flow cytometry in the neoplastic cells from 80 patients with CD5+ B-cell lymphoproliferative disorders. The D cyclins were expressed differentially in chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), and mantle cell lymphoma (MCL) with strong staining of cyclin D1 and D2 in MCL, strong staining of cyclin D1 but weak staining of cyclin D2 in 4 of 5 PLLs, and low-level staining for both cyclins in most CLLs. No correlation between cyclin D1 and D2 and growth rates or CD38 expression was observed. However, cyclin D1 levels were significantly higher in ZAP-70+ CLL cases, although no association between ZAP-70 and cyclin D2 was detected. The results indicate that flow cytometric analysis of D cyclins may help in classification of CD5+ B-cell lymphoproliferative disorders.

Topics: ADP-ribosyl Cyclase 1; CD5 Antigens; Cell Proliferation; Cyclin D1; Cyclin D2; Cyclins; Flow Cytometry; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Prolymphocytic; Lymphoma, Mantle-Cell; Phosphatidylinositol 3-Kinases; ZAP-70 Protein-Tyrosine Kinase

We present two cases of small B-cell lymphomas of particular diagnostic interest because the histological patterns were at variance with their immunophenotype. One of these lymphomas, involving the gallbladder and duodenum, showed a marginal zone lymphoma-like (MALT type) pattern of cellular infiltration with CD5 negativity but (unexpectedly) Cyclin D1 positivity. Fluorescence in situ hybridization analysis of this case was performed because of the aberrant expression of Cyclin D1, and was clearly positive for the Cyclin D1 gene translocation. The second case, occurring in a lymph node, showed the typical growth pattern of a follicular lymphoma but it had an atypical immunophenotype, namely, expression of Cyclin D1, CD10, and Bcl2 and focally Bcl6, accompanied by a lack of CD5 and CD23. The Cyclin D1 gene translocation was detected by fluorescence in situ hybridisation (FISH), whereas c-myc and Bcl2 genes translocation were absent. Numerical chromosomal changes, which were visualized for chromosomes 8, 11, and 18 could be correlated to the aberrant immunoprofile. In this context, we discuss the diagnostic value of Cyclin D1, CD5, CD23, CD10, Bcl6 markers revealed by immunohistochemistry, as well as the significance of detection by FISH of chromosomal translocations such as t(11;14) and t(14;18). The question still remains as to whether such cases should be designated as specific lymphoma entities or reported as unclassifiable and the chromosome aberration reported.

Topics: Aged; B-Lymphocytes; Biomarkers, Tumor; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Chromosomes, Human, Pair 18; Chromosomes, Human, Pair 8; Cyclin D1; Female; Humans; Immunophenotyping; In Situ Hybridization, Fluorescence; Leukemia, Lymphocytic, Chronic, B-Cell; Lymph Nodes; Male; Middle Aged; Translocation, Genetic

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and mantle cell lymphoma usually are distinctly different in regard to clinical presentation, morphology, immunophenotype, and molecular/genetic findings. In spite of this, select cases may show overlapping characteristics and represent a diagnostic challenge. Cyclin D1 immunohistochemical staining is usually envisioned as a definitive method for resolving this differential diagnosis, with positivity supporting a diagnosis of mantle cell lymphoma. We report a case involving a 58-year-old man with a diagnosis of CLL/SLL for several years. A lymph node excision was performed after increased adenopathy was noted in the cervical region. The excised lymph node showed typical morphologic findings of CLL/SLL, including the presence of characteristic proliferation centers. Cyclin D1 staining, using 3 different antibodies, was present in scattered prolymphocytes and paraimmunoblasts, mostly within proliferation centers. Fluorescence in situ hybridization and conventional cytogenetics demonstrated trisomy 12 and an absence of t(11;14) in lymph node tissue. Focal cyclin D1 expression by immunohistochemistry in nodal CLL/SLL is quite unusual and is discussed as a potential diagnostic pitfall.

Topics: Chromosomes, Human, Pair 12; Cyclin D1; Diagnosis, Differential; Humans; Immunophenotyping; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Mantle-Cell; Male; Middle Aged; Trisomy

Translocations involving IGH are common in some lymphoid malignancies but are believed to be rare in chronic lymphocytic leukaemia (CLL). To study the clinical utility of fluorescence in situ hybridization (FISH) for IGH translocations, we reviewed 1032 patients with a presumptive diagnosis of CLL. Seventy-six (7%) patients had IGH translocations. Pathology and clinical data were available for the 24 patients evaluated at the Mayo Clinic. Ten (42%) patients had IGH/cyclin D1 fusion and were diagnosed with mantle cell lymphoma (MCL). The immunophenotype was typical of MCL in three of these patients and atypical for MCL in seven patients. One patient had biclonal disease with typical MCL and CLL with IGH/BCL-2. Eleven (46%) patients had IGH/BCL-2 fusion including the patient with biclonal disease. Two of these patients had leukaemic phase follicular lymphoma and nine patients had CLL. The median progression-free survival of patients with CLL and IGH/BCL-2 translocation was 20.6 months. The two patients with IGH/BCL-3 fusion (one of these also had IGH/BCL-11a) had rapid disease progression. The IGH partner gene was not identified in two patients. We conclude that use of an IGH probe in FISH analysis of monoclonal B-cell lymphocytosis improves diagnostic precision and could have prognostic value in patients with CLL.

Topics: B-Cell Lymphoma 3 Protein; Cyclin D1; Diagnosis, Differential; Flow Cytometry; Genes, bcl-2; Genes, Immunoglobulin; Humans; Immunoglobulin Heavy Chains; Immunophenotyping; In Situ Hybridization, Fluorescence; Interphase; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Lymphoma, Mantle-Cell; Oligonucleotide Probes; Proto-Oncogene Proteins; Transcription Factors; Translocation, Genetic

The differential diagnosis of certain B CD5+ lymphoproliferative processes, such as mantle cell lymphoma (MCL) and atypical chronic lymphocytic leukemia (ACLL), is difficult. The aim of this study was to correlate morphological findings, cyclin D1 (cD1) detection by immunohistochemistry (IHC) and immunophenotype by flow cytometry (FC) with the results obtained by molecular biology in this type of neoplasias. We analyzed 20 samples classified as B CD5+ lymphoproliferative processes by FC. PCR was used for t(11;14) bcl-1/IgH determination. Histopathological and IHC studies for cD1 were done in 14 cases. Twelve cases were diagnosed as MCL, with positive cD1 in 5 (5/9), five as ACLL and three as B lymphoproliferative process. PCR revealed t(11;14) in 6/12 MCL and negative results in the other groups (0/8). Molecular biology evidenced translocation in 4/5 MCL positive for cD1 with IHC. The presence of translocation could be demonstrated by IHC and PCR in 7/12 MCL: 4 with both techniques, 2 with PCR alone, and 1 with IHC alone. These findings show a significant association between cD1 by IHC and bcl-1/ IgH gene detection by PCR, which implies that both techniques are complementary for MCL typing.

Topics: Adult; Aged; Bone Marrow; CD5 Antigens; Cyclin D1; Diagnosis, Differential; Female; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymph Nodes; Lymphoma, Mantle-Cell; Male; Middle Aged; Spleen

Cyclins are very important components of the cell cycle machinery because their levels regulate cell proliferation. They have also been found to be prognostic factors in various cancers. We studied the expression of the positive cell cycle regulators (D cyclins) and the cell proliferation marker (Ki-67) in human acute myeloid (AML), chronic myeloid (CML), acute lymphoblastic (ALL) and chronic lymphocytic (CLL) leukemia [mainly by comparative reverse transcription polymerase chain reaction (RT-PCR)]. Both leukemic and normal cells were positive for cyclin D3 expression. Significant differences were found in the expression of cyclin D1, which was the highest in leukocytes (CD19 + ) of CLL patients whereas lower expression was found in CML, AML and ALL patients and normal bone marrow and peripheral blood leukocytes (P < 0.001). The higher expression of cyclin D1 in leukocytes of CLL patients compared to CML patients was confirmed by quantitative real-time RT-PCR with a TaqMan probe in a subset of CLL and CML patients. Differences in cyclin D1 expression between CLL and CML patients were also confirmed on protein levels by western blotting. Expression of the proliferative marker Ki-67 was high in CML, ALL and AML cells and low in CD19-positive CLL cells. The results demonstrate that the level of cyclin D1 negatively correlates with the proliferation properties of leukemic cells. We did not find any significant relationship between cyclin D1 expression in cells of CML and AML patients and their clinical outcome.

Topics: Acute Disease; Cell Proliferation; Cyclin D1; Cyclin D2; Cyclin D3; Cyclins; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Ki-67 Antigen; Leukemia; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Reverse Transcriptase Polymerase Chain Reaction

To investigate the feasibility of detecting cyclin D1 mRNA in paraffin-embedded tissues by reverse transcriptase polymerase chain reaction (RT-PCR) and competitive RT-PCR and its diagnostic and differential diagnostic significance for mantle cell lymphoma (MCL).. Paraffin-embedded samples of 36 cases of MCL, 71 cases of other small B-cell lymphomas and 20 cases of lymphoid reactive hyperplasia as control group were retrieved from archival materials. Cyclin D1 protein and its mRNA was detected by EnVision and RT-PCR and competitive RT-PCR in all samples. House-keeping gene PGK was choosen as internal control.. (1) Cyclin D1 protein was expressed in 27 of the 38 MCL (71.1%). No cyclin D1 expression was found in the control group. (2) PGK was detected in 103 of the 116 cases (88.8%) and also detected in 34 of 36 MCL cases (94.7%). (3) cyclin D1 mRNA was detected in 34 nodal mantle cell lymphoma cases by RT-PCR in paraffin-embedded tissues. The positive rate of cyclin D1 mRNA was 94.4% in mantle cell lymphomas after exclusion of the 2 cases which were negative for both cyclin D1 mRNA and PGK. cyclin D1 mRNA was not detected in other nodal small B-cell lymphomas or lymphoid reactive hyperplasia, except 1 case of B-SLL. Sequencing analysis showed that sequences were identical to cyclin D1. (4) Cyclin D1 mRNA overexpression was detected in 27 cases of nodal mantle cell lymphoma by competitive RT-PCR in paraffin-embedded tissues. The positive rate of cyclin D1 mRNA overexpression was 75.0% in mantle cell lymphomas after exclusion of 2 cases which were negative for both cyclin D1 mRNA and PGK. cyclin D1 mRNA overexpression was not detected in other nodal small B-cell lymphomas or lymphoid reactive hyperplasia.. RT-PCR and competitive RT-PCR detection of cyclin D1 mRNA overexpression could be used for the diagnosis and differential diagnosis of mantle cell lymphoma in paraffin-embedded blocks.

Topics: Cyclin D1; Diagnosis, Differential; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Follicular; Lymphoma, Mantle-Cell; Paraffin Embedding; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

B-cell chronic lymphocytic leukemia/ small lymphocytic lymphoma (B-CLL/SLL) and mantle cell lymphoma (MCL) show many overlapping morphologic and immunophenotyping features, however they have great difference in therapeutic regimens and prognosis.. Is to determine the diagnostic and prognostic role of clinico-pathologic variables, CD23 and Cyclin D1 oncoprotiens in B-SLL/CLL and MCL.. This study included 25 BCLL/ SLL cases and 25 MCL cases. All cases were carefully examined and stained using CD23 and Cyclin D1 immunostaining.. There was significant difference between BCLL/ SLL and MCL regarding several items including pattern of growth, where interfollicular pattern was restricted to B-SLL/CLL while nodular and mantle zone pattern were confined to MCL; pseudo-follicles were only present in B-CLL/SLL. Transformed cells, plasmacytoid cells, peripheral blood lymphocytosis, significant longer survival and good prognosis were statistically more prominent in favor of B-CLL/SLL. On the other hand, cell cleavage, epithelioid histiocytes, plasma cells, naked nuclei, hyalinized venules, deposited hyaline material in background and reticular fibers in addition to higher mitotic index per 20 HPF were more significantly identified in favor of MCL. CD23 was expressed as membranous pattern in 16/25 (64%) of B-CLL/SLL cases and 1/25 (4%) of MCL cases. On the other hand, Cyclin D1 was expressed as nuclear staining in 18/25 (72%) of MCL cases and only 1/25 (4%) of B-CLL/SLL cases. Regarding B-CLL/SLL, age >60 years and mitosis >or=10/20 HPF were independent prognostic factors of shorter survival by multivariate analysis. In MCL, Cyclin D1 overexpression and splenomegaly were independent prognostic factors of survival by multivariate analysis.. Cyclin D1 is not only implicated in tumor genesis of MCL, but also in progression and extension of the disease when expressed in high levels (50% cut off value) and it seems to have prognostic impact in MCL. This can be used as a basis for future therapeutic strategies targeting cell cycle regulators. This study could support the concept that Cyclin D1 and CD23 immunostaining may be reliable diagnostic tools for discrimination between B-CLL/SLL and MCL.

Topics: Biomarkers, Tumor; Cyclin D1; Diagnosis, Differential; Humans; Immunohistochemistry; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Mantle-Cell; Middle Aged; Prognosis; Receptors, IgE; Survival Analysis

B cell chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature, functionally incompetent B cells. Wnts are a large family of secreted glycoproteins involved in cell proliferation, differentiation, and oncogenesis. The classical Wnt signaling cascade inhibits the activity of the enzyme glycogen synthase kinase-3beta, augmenting beta-catenin translocation to the nucleus, and the transcription of target genes. Little is known about the potential roles of Wnt signaling in CLL. In this study, we quantified the gene expression profiles of the Wnt family, and their cognate frizzled (Fzd) receptors in primary CLL cells, and determined the role of Wnt signaling in promoting CLL cell survival. Wnt3, Wnt5b, Wnt6, Wnt10a, Wnt14, and Wnt16, as well as the Wnt receptor Fzd3, were highly expressed in CLL, compared with normal B cells. Three lines of evidence suggested that the Wnt signaling pathway was active in CLL. First, the Wnt/beta-catenin-regulated transcription factor lymphoid-enhancing factor-1, and its downstream target cyclin D1, were overexpressed in CLL. Second, a pharmacological inhibitor of glycogen synthase kinase-3 beta, SB-216763, activated beta-catenin-mediated transcription, and enhanced the survival of CLL lymphocytes. Third, Wnt/beta-catenin signaling was diminished by an analog of a nonsteroidal antiinflammatory drug (R-etodolac), at concentrations that increased apoptosis of CLL cells. Taken together, these results indicate that Wnt signaling genes are overexpressed and are active in CLL. Uncontrolled Wnt signaling may contribute to the defect in apoptosis that characterizes this malignancy.

Topics: B-Lymphocytes; beta Catenin; Cell Survival; Cells, Cultured; Cyclin D1; Cytoskeletal Proteins; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Indoles; Leukemia, Lymphocytic, Chronic, B-Cell; Leukocytes, Mononuclear; Maleimides; Multigene Family; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Reference Values; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Trans-Activators; Transcription Factors; Wnt Proteins; Zebrafish Proteins

We developed a real-time, quantitative, reverse transcription PCR assay for cyclin D1 (CCND1) expression to aid in the diagnosis of mantle cell lymphoma (MCL). The diagnosis of MCL can be problematic, and existing CCND1 expression assays show a lack of specificity, with elevated expression also detected in other lymphoproliferative disorders. We postulated that evaluating CCND1 expression relative to CCND3 expression by quantitative PCR could offer an improved specificity over an evaluation of CCND1 alone. This method quantitates both CCND1 and CCND3, each normalized to a housekeeping gene (GADPH), using the 5'-exonuclease technique. We analyzed 107 clinical specimens: MCL (17), chronic lymphocytic leukemias (CLL) (10), other non-MCL hematolymphoid disorders (41), non-malignant tissues with an epithelial component (7) and other normal samples (32). This method correctly identified 16 of 17 MCLs, and there were no false positives among any of the other diagnostic groups tested including CLL. CLL presents the major diagnostic dilemma at this institution when diagnosing MCL. Sensitivity studies showed that this method could detect an elevated CCND1/CCND3 ratio when the tumor infiltrate is at least 10% of the cells. We compared the specificity of CCND1 expression alone against the CCND1/CCND3 ratio to demonstrate the increased specificity for the latter. We conclude that the CCND1/CCND3 ratio is a sensitive and specific test for the diagnosis of MCL.

Topics: Cyclin D1; Cyclin D3; Cyclins; Hematologic Neoplasms; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Mantle-Cell; Lymphoproliferative Disorders; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity

Rabbit monoclonal antibody (MAb), which has become available only recently, theoretically combines the advantage of the high affinity attributable to its rabbit origin and the high specificity due to its monoclonal nature. Since immunohistochemical demonstration of cyclin D1 is notoriously difficult, this study aims to assess whether a newly available rabbit MAb against cyclin D1 (SP4) can improve the consistency of immunostaining, especially for the diagnosis of mantle cell lymphoma (MCL). A total of 150 cases of lymphoproliferative lesions, including 30 cases of MCL, histologic mimickers of MCL, and various types of lymphomas and leukemias, were studied. Immunostaining was performed on formalin-fixed, paraffin-embedded tissue sections using a labeled streptavidin-biotin peroxidase system in an automated immunostainer. All cases of MCL expressed cyclin D1, with a higher median staining score (8 out of a maximum of 12) compared with mouse MAb DCS-6 (score 4). In addition, 2 of 15 cases of B-cell chronic lymphocytic leukemia (B-CLL), 3 of 12 cases of multiple myeloma, and 2 of 5 cases of hairy cell leukemia were also positive. Comparable staining results could also be achieved by an optimized manual staining protocol. This study thus confirms the superior performance of the rabbit MAb SP4, which should permit consistent immunostaining for cyclin D1 to be readily achieved. The value of cyclin D1 immunohistochemistry in the differential diagnosis of MCL from other low-grade B-cell lymphomas is also affirmed, but with the caveat that rare cases of B-CLL can also be cyclin D1 positive.

Topics: Animals; Antibodies, Monoclonal; Biotin; Cyclin D1; Diagnosis, Differential; Humans; Immunohistochemistry; Leukemia, Hairy Cell; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Mantle-Cell; Rabbits; Staining and Labeling; Streptavidin

Classical t(11;14)(q13;q32) involving IGH-CCND1 is typically associated with aggressive CD5-positive mantle cell lymphoma (MCL). Recently, we identified the IGK variant of this translocation, t(2;11)(p11;q13), in three patients with a leukemic small-cell B-non-Hodgkin lymphoma. In all cases, rearrangements of the IGK and CCND1 genes were demonstrated by fluorescence in situ hybridization. Moreover, we mapped the 11q13 breakpoint of this variant translocation in the 3' region of CCND1 which contrasts with the 5' breakpoints in a standard t(11;14)(q13;q32). Expression of cyclin D1 was shown in two cases analyzed either at diagnosis or during disease progression. All three patients were asymptomatic at presentation and no initial therapy was required. One patient died of a progressive disease 58 months from diagnosis, and two patients showed stable disease after 12 months of follow-up. In two analyzed cases, mutated IGVH genes were identified. Our findings indicate that variant t(2;11)(p11;q13) does not typify a classical MCL but possibly a more indolent leukemic lymphoma originating from an antigen experienced (mutated) B cell.

Topics: Adult; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 2; Cyclin D1; Disease Progression; DNA, Neoplasm; Female; Gene Rearrangement, B-Lymphocyte, Heavy Chain; Genetic Variation; Humans; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Immunoglobulins; In Situ Hybridization, Fluorescence; Karyotyping; Leukemia; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Mantle-Cell; Lymphoma, Non-Hodgkin; Male; Middle Aged; Mutation; Neoplasm Recurrence, Local; Translocation, Genetic

Studies to investigate signal transduction pathways that support viability and prevent apoptosis of chronic lymphocytic leukemia cells (CLL) were initiated as a result of microarray cDNA analyses which revealed expression of genes whose products regulate cell cycle progression. Immunoblots revealed translation of several genes including caspases, cyclin D1, and the PI3-kinase dependent, survival kinase, Akt. Akt was found to be activated. Inhibition of PI3-kinase with specific inhibitor, LY294002, led to the induction of apoptosis that was caspase 8 dependent, but independent of Akt as LY294002 did not depress a high basal level of Akt activity found in CLL cells. Phosphorylation of Akt was maintained, enzymatic activity undiminished, and phosphorylation of substrates sustained. Caspases, however were activated, PARP cleaved and DNA fragmented. Caspase inhibitors revealed that initiator caspase 8 was required for classic apoptosis when PI3-kinase was inhibited, and specific activity assays demonstrated its early activation. GSK-3beta a kinase regulated via PI3-kinase dependent, down-stream kinases, was responsible for regulating cyclin D1 levels in CLL cells, but neither GSK-3beta nor calpain was responsible for induction of apoptosis, or activation of executioner caspase 3, following LY294002 treatment. PI3-kinase mediated protection against caspase activation in CLL B-cells therefore is not mediated through classic Akt survival pathways. The data further support the hypothesis that signal transducing, membrane associated receptors triggered by extrinsic factors, maintain CLL leukemic B-cell survival in vivo by preventing caspase activation.

Topics: Apoptosis; Calpain; Caspase 8; Caspase Inhibitors; Caspases; Cell Cycle; Cell Survival; Chromones; Cyclin D1; Enzyme Activation; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Tumor Cells, Cultured

Topics: Aged; Aged, 80 and over; Antigens, CD; Antigens, CD20; Biopsy; CD79 Antigens; Cyclin D1; Female; Fingers; Humans; Japan; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemic Infiltration; Lymphocytes; Receptors, Antigen, B-Cell; Receptors, IgE

Mantle cell lymphoma is characterized by a t(11;14)(q13;q32) translocation resulting in cyclin D1 protein overexpression. Immunohistochemical detection of the latter, therefore, is a useful marker for the diagnosis of mantle cell lymphoma. Nevertheless, interpretation of results is often hampered by the weak immunoreactivity obtained with routine detection techniques. This problem can be overcome by resorting to highly sensitive catalyzed signal amplification methods based on peroxidase-catalyzed deposition of a biotinylated phenolic compound. The present study compares the results obtained with catalyzed signal amplification, labeled streptavidin biotin, and dextran polymeric conjugate (EnVision+) techniques in cyclin D1 demonstration in mantle cell lymphoma. The study was performed on formalin-fixed, paraffin-embedded archival tissue from 20 mantle cell lymphoma cases. Ten cases of small lymphocytic lymphoma and 10 instances of follicular center cell lymphoma were used as controls. Antigen retrieval was done by autoclaving under controlled pressure (2 bar) and temperature (120 degrees C) conditions. The best results were obtained after 1 minute of exposure with catalyzed signal amplification and after 6 minutes with other detection systems. Regarding cyclin D1 expression in mantle cell lymphoma cases, 17 (85%) were weakly positive and 3 (15%), moderately positive with labeled streptavidin biotin, whereas 15 (75%) were weakly positive and 5 (25%) moderately positive with EnVision+. In contrast, all 20 mantle cell lymphoma cases were strongly cyclin D1 positive with catalyzed signal amplification. No evidence of cyclin D1 immunostaining was obtained in any of the small lymphocytic lymphoma and follicular center cell lymphoma instances with any of the three methods used. In conclusion, catalyzed signal amplification methods provide a very useful tool for cyclin D1 demonstration in cases in which other immunohistochemical techniques yield inconclusive results.

Topics: Biomarkers, Tumor; Catalysis; Cyclin D1; Humans; Immunoenzyme Techniques; Immunohistochemistry; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Follicular; Lymphoma, Mantle-Cell; Peroxidase

We studied Cyclin D1 (CyD1) and CD23 mRNA expression with real-time quantitative reverse transcription polymerase chain reaction (RQ-PCR) method. CyD1 expression in peripheral blood of seven mantle cell lymphoma (MCL) patients was found to be 1305.4 times higher than in 24 B-chronic lymphocytic leukemia (CLL) patients. CD23 expression in CLL was found to be 54.8 times higher than in MCL. These differences were statistically significant, and no overlap was found in CyD1 expression intensities between MCL and CLL. RQ-PCR allows rapid, simple and accurate quantification of CyD1 and CD23 expression, even from small samples, and is thus useful for the diagnosis of MCL and CLL.

Topics: Adult; Aged; Aged, 80 and over; CD5 Antigens; Cyclin D1; Female; Gene Expression Regulation, Leukemic; Gene Expression Regulation, Neoplastic; Genes, Immunoglobulin; Humans; Immunoglobulin Heavy Chains; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Mantle-Cell; Male; Middle Aged; Neoplasm Proteins; Oncogene Proteins, Fusion; Receptors, IgE; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity

Overexpression of cyclin D1 mRNA and protein as a result of the chromosomal translocation t(11;14)(q13;q32) is a highly specific molecular marker of mantle cell lymphoma, but cyclin D1 dysregulation can also be found in other B-cell neoplasias. The aim of the study was to develop a precise and reliable tool for quantitation of cyclin D1 mRNA suitable for archival clinical specimens.. A real-time reverse transcription-PCR (RT-PCR) assay was used to quantitate cyclin D1 mRNA copy numbers. Using 2000 microdissected cells as template, 104 formalin-fixed, paraffin-embedded lymph node, spleen, and decalcified bone marrow biopsies from a panel of 95 cases of B-cell non-Hodgkin's lymphomas (B-NHLs) were analyzed. In addition, cyclin D1 protein expression was assessed by immunohistochemistry.. Strong cyclin D1 mRNA overexpression was detected in mantle cell lymphomas (23 of 23), hairy cell leukemias (5 of 19), and multiple myelomas (7 of 23) with particularly high levels in 2 of the latter cases. Intermediate transcript levels were found in 5 of 23 multiple myelomas and 7 of 19 hairy cell leukemias. B-cell chronic lymphocytic leukemias (10 of 10), follicular lymphomas (9 of 9), mucosa-associated lymphoid tissue lymphomas (5 of 5) and reactive lymphoid tissues with the exception of normal spleen had no or very low cyclin D1 expression. In comparison with real-time RT-PCR, immunohistochemistry showed a lower level of sensitivity, more variability, and did not allow accurate quantitation.. Real-time RT-PCR for cyclin D1 mRNA is an excellent tool for the differential diagnosis of B-NHLs and, in combination with microdissection, a powerful approach for retrospective trials using archival clinical specimens as tissue source. Furthermore, real-time RT-PCR may help to identify subgroups of B-NHLs according to cyclin D1 mRNA copy numbers and to investigate the possible influence of different chromosomal breakpoints on cyclin D1 expression.

Topics: Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Computer Systems; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Leukemia, B-Cell; Leukemia, Hairy Cell; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoid Tissue; Lymphoma, B-Cell; Lymphoma, B-Cell, Marginal Zone; Lymphoma, Follicular; Lymphoma, Mantle-Cell; Multiple Myeloma; Neoplasm Proteins; Paraffin Embedding; Pseudolymphoma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Translocation, Genetic

Cyclin D1 overexpression is a valuable marker for the diagnosis of mantle cell lymphoma (MCL). We used a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) method to quantify levels of cyclin D1, CD20, and cyclophilin A mRNA in manually microdissected, paraffin-embedded tissue sections using an ABI 7700 qRT-PCR system. The study group included 21 cases of MCL and 37 cases of other types of B-cell non-Hodgkin's lymphoma. Cyclin D1 mRNA copy number was normalized to CD20 and cyclophilin A mRNA and evaluated statistically by analysis of variance. The relative cyclin D1 levels were similar whether normalized to CD20 or cyclophilin A, indicating that CD20 levels are stable and can be used as a B-cell-specific normalizer. Statistically significant differences were found in the median levels of cyclin D1 mRNA (expressed as % CD20 mRNA) among cases of MCL (87.6), small lymphocytic lymphoma (9.9), follicular lymphoma (2.4), diffuse large B-cell lymphoma (5.9), marginal zone B-cell lymphoma (39.8), and Burkitt lymphoma (7.1) (P < 0.05). We conclude that qRT-PCR can be used to quantify cyclin D1 mRNA levels in archival tissue sections. Normalization of cyclin D1 to a B-cell-specific marker more accurately reflects overexpression by MCL than other methods that normalize using constitutively expressed mRNA species.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, CD20; Archives; Biomarkers, Tumor; Burkitt Lymphoma; Cyclin D1; Cyclophilin A; DNA Primers; Female; Humans; Immunoenzyme Techniques; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Lymphoma, Large B-Cell, Diffuse; Lymphoma, Mantle-Cell; Lymphoma, Non-Hodgkin; Male; Middle Aged; Paraffin Embedding; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm

Six cases of non-Hodgkin B-cell lymphoma that mimicked either chronic lymphocytic leukemia (CLL) or a CLL variant at presentation are reported. The patients ranged from 54 to 89 years and included three females and three males. All six patients had prominent peripheral blood lymphocytosis at presentation; the initial morphologic impression was CLL in three cases, CLL/prolymphocytic leukemia (PLL) in two cases, and PLL in one. Five patients had bone marrow biopsies; each showed a lymphoid infiltrate in a focally random, interstitial, and/or diffuse pattern. Flow cytometric immunophenotyping showed CD20-positive B cells with surface immunoglobulin (Ig) light chain restriction in all six patients. The five cases resembling CLL or CLL/PLL had at least a subset of CD5-positive B cells, whereas CD5 was absent in the one case that resembled PLL. CD23 was positive in three of the four cases studied that resembled CLL or CLL/PLL; CD79b was positive in three, FMC7 was positive in two, and surface Ig and CD20 were brightly positive in three. A t(11;14) (q13;q32) was found in four cases that resembled CLL or CLL/PLL; they were subsequently diagnosed as mantle cell lymphoma. The remaining two cases mimicking CLL or PLL were diagnosed as lymphomas of follicle center origin with leukemic phase based on the presence of t(14;18) (q32;q21). Thus although the morphology of these six cases resembled CLL or variants, and immunophenotyping by flow cytometry showed overlapping features, genetic studies enabled distinction of these leukemic non-Hodgkin lymphoma from chronic lymphocytic leukemia or variants.

Topics: Aged; Aged, 80 and over; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Chromosomes, Human, Pair 18; Cyclin D1; Diagnosis, Differential; Female; Flow Cytometry; Humans; Immunohistochemistry; Immunophenotyping; In Situ Hybridization, Fluorescence; Karyotyping; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Male; Middle Aged; Translocation, Genetic

To describe and revise a flow cytometric assay for evaluating cyclin D1 overexpression in B cell lymphoproliferative disorders (B-LPDs).. Cyclin D1 expression was evaluated in 11 healthy controls and 51 patients with B-LPD by flow cytometry using the 5D4 monoclonal antibody. In 25 cases, experiments were repeated up to four times with mononuclear cells (MNC) fixed in ethanol for 1-120 days to evaluate the consistency of cyclin D1 expression. Flow cytometry results were compared with fluorescence in situ hybridisation (FISH) for the t(11;14) translocation in 19 patients and with immunohistochemistry (IHC) using the DCS-6 monoclonal antibody in nine patients.. A mean fluorescence intensity ratio (MFIR) of 4.8 was defined as the cut off point for positivity based on cyclin D1 expression in healthy controls (mean + 3 SD). Ten patients overexpressed cyclin D1 by flow cytometry. These included five of eight patients with mantle cell lymphoma, four of 19 with chronic lymphocytic leukaemia, and one with follicular lymphoma. MFIR in the repeat experiments differed less than 25% in 20 of 25 patients and in no cases did it cross the cut off point. There was a good correlation between cyclin D1 expression by flow cytometry and FISH for t(11;14) in 15 of 19 patients and six of nine had concordant results with flow cytometry, FISH, and IHC.. Cyclin D1 expression remains fairly stable once MNC are fixed in ethanol and the flow cytometric assay can be used for the routine screening of B-LPD. Further comparisons between flow cytometry, IHC, and FISH may be needed to ascertain the diagnostic value of the flow cytometric assay.

Topics: B-Lymphocytes; Biomarkers, Tumor; Cyclin D1; Female; Flow Cytometry; Humans; Immunoenzyme Techniques; In Situ Hybridization, Fluorescence; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Lymphoma, Mantle-Cell; Lymphoproliferative Disorders; Male; Neoplasm Proteins; Reproducibility of Results

B-CLL cells are arrested in G0/early G1 phase of the cell cycle and are characterized by a marked hyporesponsiveness towards a variety of polyclonal B cell activators. We have previously demonstrated that costimulation with CpG-ODN and IL-2 can overcome this proliferative defect. Cyclin D3 is the principal D-type cyclin which mediates G1 progression in normal B cells, but in B-CLL cells both cyclin D2 and cyclin D3, were strongly upregulated upon stimulation. Both cyclins were associated with cdk4 but not with cdk6, which is the catalytic partner of D-type cyclins in normal B cells. Moreover, immune complexes consisting of cyclin D2 and cdk4 or cyclin D3 and cdk4 were both functional and phosphorylated the RB protein in vitro. The cell cycle inhibitor p27 plays a pivotal role in cell cycle progression of B lymphocytes and has been shown to be overexpressed in B-CLL cells. P27 was rapidly downregulated in B-CLL cells even when stimulated with a non-CpG-ODN or IL-2 alone, while only moderate regulation could be observed in normal B cells. Taken together, our findings demonstrate that regulation of early cell cycle progression differs between B-CLL cells and normal B cells. These findings do not only contribute to the understanding of B-CLL pathophysiology, but might ultimately lead to the identification of new therapeutic targets.

Topics: Apoptosis; B-Lymphocytes; Cell Cycle; Cell Cycle Proteins; Cyclin D1; Cyclin D2; Cyclin D3; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; DNA Primers; Drug Combinations; Flow Cytometry; Humans; Immunoblotting; Interleukin-2; Leukemia, Lymphocytic, Chronic, B-Cell; Oligodeoxyribonucleotides; Phosphorylation; Precipitin Tests; Proto-Oncogene Proteins; Retinoblastoma Protein; Thymidine; Tumor Cells, Cultured; Tumor Suppressor Proteins

We have studied the expression of MIB-1 and prognosis in cyclin D1(CyD1)+ and CyD1- mantle cell lymphoma (MCL), and compared them to B-CLL/SLL. All cases were assigned to four groups by immunoreactivity and primary sites: (1) CyD1+ nodal MCL, 11 cases: (2) CyD1+ extranodal MCL (multiple lymphomatous polyposis, (MLP)) three cases: (3) CyD1- nodal MCL, three cases: and (4) CyD1- B-CLL/SLL, seven cases. The average of MIB-1 labeling indexes of the four groups were 30.66, 8.70, 9.30 and 4.66, respectively. The CyD1- group consisting of nodal MCL and CLL/SLL had a significantly longer median survival time (69 months) than the CyD1+ group consisting of nodal MCL and MLP (22 months, P = 0.01). These data indicate that CyD1- nodal MCL may show a lower MIB-1 labeling index, and has a better prognosis, than CyD1+ nodal MCL. In addition, a large difference in the average of MIB-1 labeling indexes between nodal MCL and MLP in the CyD1+ group was found.

Topics: Aged; Aged, 80 and over; Biomarkers; Chronic Disease; Cyclin D1; Disease Progression; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Lymphoma, Mantle-Cell; Male; Middle Aged; Neoplasm Proteins; Prognosis; Retrospective Studies; Survival Analysis

To investigate the role of the cell cycle regulators p21(Waf1), p27(Kip1), retinoblastoma (Rb), and cyclin D1 in Richter's transformation of chronic lymphocytic leukemia (CLL), we analyzed 19 CLL and eight Richter's syndrome (RS) tumors, previously characterized for p53 and ARF/INK4a abnormalities. p21(Waf1)immunohistochemical expression was negative in 12 of 15 CLL (80%), whereas it was moderate or strong in three of seven RS (43%). p21(Waf1) gene was in germline configuration in all the tumors analyzed. Four immunohistochemical patterns of p53 and p21(Waf1) expression were observed: (1) p53-/p21- in 10 of 15 CLL (67%), but only in two of six RS (33%); (2) p53+/p21+ in three CLL (20%) and two RS (33%); (3) p53-/p21+ in one RS; and (4) p53++/p21- in two CLL and one RS. Two p53+/p21+ CLL evolved into RS. p53 mutations clustered around the p53++/p21- (two CLL and one RS) and p53-/p21- (one CLL and one RS) tumors. While the majority of CLL displayed strong p27 immunoreactivity, RS tumors were constantly p27-negative. p27(Kip1) gene was in germline configuration in all the tumors analyzed. Most CLL cases were negative for Rb expression. In contrast, all RS exhibited strong Rb expression. Cyclin D1 overexpression was only detected in one CLL evolving into RS and one RS. In conclusion, a p53+/p21- immunohistochemical pattern is shown exclusively by p53-mutated CLL/RS. Additionally, our results suggest a possible implication of moderate/strong p21(Waf1) expression, loss of p27 expression, and cyclin D1 overexpression in the Richter's transformation of CLL.

Topics: Adult; Aged; Cell Cycle; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Female; Genes, p53; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Large B-Cell, Diffuse; Male; Middle Aged; Mutation; Retinoblastoma Protein; Tumor Suppressor Proteins

We studied 20 cases of mature B-cell leukemia with more than 55% prolymphocytes in peripheral blood or bone marrow, fulfilling the French-American-British criteria for B-cell prolymphocytic leukemia (PLL). Cases segregated into 3 groups: de novo PLL, 6; PLL occurring in patients with a previous well-established diagnosis of chronic lymphocytic leukemia (PLL-HxCLL), 10; and t(11;14)(q13;q32)-positive neoplasms, 4. All cases expressed monotypic immunoglobulin light chain, and most were positive for CD5. All t(11;14)-positive neoplasms were CD23- and uniquely positive for cyclin D1. Cytogenetic abnormalities were present in 19; in all 19, the karyotype was complex, indicating clonal evolution and genomic instability. The most frequent cytogenetic abnormality in de novo PLL involved chromosome 7 in 4 cases. Trisomy 12 or add(12p) was present in 4 cases of PLL-HxCLL. We conclude that mature B-cell leukemias with more than 55% prolymphocytes are a heterogeneous group that includes t(11;14)-positive neoplasms, which we suggest are best classified as mantle cell lymphoma. We also suggest that prolymphocytic morphologic features are a common end-stage of transformation for several B-cell neoplasms.

Topics: Aged; Aged, 80 and over; Bone Marrow Cells; CD5 Antigens; Cell Separation; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 13; Chromosomes, Human, Pair 7; Cyclin D1; Female; Flow Cytometry; Humans; Immunohistochemistry; Leukemia, B-Cell; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocytes; Lymphoma, Mantle-Cell; Male; Middle Aged; Prognosis; Receptors, IgE; Trisomy

The cyclin D1 gene, CCND1, is alternatively spliced to produce transcripts [a] and [b] in a manner apparently modulated by a polymorphism (A/G) at codon 241. Studies have indicated that the polymorphism can affect the prognosis of patients with different types of solid tumors. This study aimed to determine the relative levels of transcripts [a] and [b] in normal and malignant peripheral blood mononuclear cells (PBMNC), and to investigate whether these were influenced by the polymorphism. The impact of the polymorphism on the survival of a group of mantle cell lymphoma (MCL) patients was also to be studied.. The polymorphism was genotyped, using restriction fragment length polymorphism analysis, in 74 patients (42 MCL, 19 chronic lymphocytic leukemia, 13 normal controls) and the relative level of transcripts [a] and [b] determined using a competitive reverse transcription polymerase chain reaction method. Kaplan-Meier survival curves and the log-rank test were used to analyze the survival data.. Of the cases genotyped, 39 were heterozygous for the polymorphism, 24 homozygous G and 11 homozygous A. Both transcripts [a] and [b] were expressed in normal PBMNC and malignant lymphocytes, with the polymorphism affecting their relative levels. Neither the predominant transcript, nor genotype, significantly influenced survival of the MCL patients studied.. Contrary to previous reports, patients who were homozygous A at the polymorphism produced more transcript [a] whilst homozygous G patients had more transcript [b]. In the small cohort studied, the polymorphism did not appear to affect the prognosis of the patients with MCL.

Topics: Adult; Aged; Aged, 80 and over; Alternative Splicing; Case-Control Studies; Cyclin D1; Female; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocytes; Lymphoma, Mantle-Cell; Male; Middle Aged; Polymorphism, Single Nucleotide; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Analysis

The distinction between mantle cell lymphoma (MCL) and other small B-cell non-Hodgkin lymphomas (NHL) is important because MCL has a more aggressive clinical course. In bone marrow (BM) biopsy specimens, this distinction can be particularly difficult. Although cyclin D1 immunostaining and molecular detection of the t(11;14) translocation are highly specific markers for MCL, they fail to detect a proportion of cases. We have recently described that MCL typically lacks detectable expression of the cyclin-dependent kinase inhibitor p27(kip1) protein by immunostaining, which is expressed at high levels in most small B-cell NHL inversely correlated to the proliferation rate. We therefore examined whether p27(kip1) immunostaining could be a useful adjunct for the differential diagnosis of small B-cell NHL infiltrates in the BM. Trephine BM biopsy specimens of 96 patients, including well-characterized MCL (19 cases), B-cell chronic lymphocytic leukemia (27 cases), follicular lymphoma (18 cases), hairy cell leukemia (22 cases), and marginal zone lymphoma (10 cases) as well as 10 reactive BM, including five with benign lymphoid aggregates were investigated. In addition, the presence of a t(11;14) translocation involving the major translocation cluster was studied by PCR in all MCL. All cases of B-cell chronic lymphocytic leukemia, follicular lymphoma, and marginal zone lymphoma revealed a strong p27(kip1) nuclear staining in the majority of neoplastic cells. Fourteen (78%) cases of MCL were p27(kip1)-negative in the tumor cells, whereas four cases revealed a weak nuclear positivity. Seventeen (77%) cases of hairy cell leukemia were also either completely negative for p27(kip1) or showed a faint positive staining in a minority of the neoplastic cells. Nine of 19 cases (47%) of MCL showed a bcl1 rearrangement involving the major translocation cluster region. These findings demonstrate that p27(kip1) immunostaining is a valuable additional marker for the differential diagnosis of small B-cell NHL infiltrates in BM biopsies. The reduction or lack of p27(kip1) protein expression in MCL, as well as in hairy cell leukemia, might be an important event in the pathogenesis of these disorders.

Topics: Biopsy; Bone Marrow; CD3 Complex; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Diagnosis, Differential; DNA, Neoplasm; Gene Rearrangement; Humans; Immunohistochemistry; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Lymphoma, Mantle-Cell; Lymphoma, Non-Hodgkin; Tumor Suppressor Proteins

We compared the features of 17 cases of atypical chronic lymphocytic leukemia (aCLL) with those of a clinical control group of 24 cases of CLL. Quantitative flow cytometric data, available for 12 cases, were compared with an immunophenotypic control group of 58 cases using a relative fluorescence indexfor CD5, CD23, CD79b, and surface immunoglobulin light chain (sIg). Compared with the clinical control group, patients with aCLL had a higher mean WBC count and a lower platelet count. Patients with aCLL had a significantly higher probability of disease progression. Compared with an immunophenotypic control group of 58 CLL cases, 12 cases of aCLL demonstrated significantly higher expression of CD23. There was no significant difference in expression of sIg, CD79b, or CD5 between the groups. CD38 expression was noted in only 1 (9%) of 11 tested cases; 2 (18%) of 11 cases had trisomy 12. aCLL can be distinguished from typical CLL morphologically, clinically, and immunophenotypically. Atypical morphologic features in CLL seem to be a marker of aggressive clinical behavior.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD; Cyclin D1; Disease Progression; DNA Primers; DNA, Neoplasm; Female; Flow Cytometry; Humans; Immunoenzyme Techniques; Immunoglobulin Light Chains; Immunophenotyping; In Situ Hybridization, Fluorescence; Karyotyping; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Prolymphocytic; Male; Middle Aged; Polymerase Chain Reaction

Assessment of the expression of antigens CD5, CD10 and CD23 can be of value in the differential diagnosis of small B-cell lymphoma. Correct subclassification is important since optimal treatment regimes differ between the subtypes. The aim of this study was to generate monoclonal antibodies recognizing these antigens in paraffin-embedded tissue and to assess their efficacy using a panel of cases of small B-cell lymphoma of various subtypes.. For each antibody synthetic recombinant protein and conventional murine hybridoma technology was employed. Monoclonal antibodies effective in formalin-fixed, paraffin-embedded tissue were successfully generated, designated NCL-CD5-4C7, NCL-CD10-270 and NCL-CD23-1B12, respectively. A series of 58 cases of small B-cell lymphoma including examples of each subtype (lymphocytic, follicle centre cell, mantle cell, marginal zone and lymphoplasmacytoid) was assembled and immunostaining for the respective antigens carried out using the monoclonal antibodies produced. Our results indicate that the antibodies are specific for their respective antigens and give the predicted phenotypic profile in the small B-cell lymphoma subtypes.. These novel monoclonal antibodies may be of value in routine diagnostic practice.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Blotting, Western; CD5 Antigens; Cyclin D1; Fixatives; Formaldehyde; Humans; Immunohistochemistry; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Lymphoma, Mantle-Cell; Mice; Neprilysin; Paraffin Embedding; Receptors, IgE; Tissue Fixation

Cyclin D1 (CyD1)/BCL1 (PRAD1) is expressed at high levels in almost all cases of mantle cell leukemia/lymphoma (MCL) and in rare cases of chronic lymphocytic leukemia (CLL). The CyD1/BCL1 protein plays an important role in the progression of cells through the G1 phase of cell cycle. Most of the CyD1/BCL1 protein expression studies are performed using immunohistochemistry. We used a sensitive solid-phase radioimmunoassay (RIA) to quantify CyD1 protein expression in 199 patients with CLL. Of these 137 patients were previously untreated with the rest having had standard chemotherapeutic regimens including alkylating agents and fludarabine before being referred to our center. Median white cell count in these patients was 49x10(3) /microl (range 3.0-438.5x10(3)/microl), hemoglobin level 13.1 g/dl (range 5.2-17.3 g/dl), platelet count 157x10(3) /microl (range 10-377x10(3) /microl), age 58 (range 26-89), and beta2-microglobulin 2.75 mg/dl (range 1.1-14.3). The median radioactivity (CPM) of mononuclear cells obtained from 56 normal individuals was assigned a value of 1. There was no significant variation in CyD1 levels among normal individuals (SD=0. 12). While most CyD1 levels in MCL varied from 6.5 to 15.6, the median CyD1/BCL1 in CLL was 1.4 with 75th percentile under 2.12. Rare CLL cases (3.5%) showed levels between 4 and 8.83. When divided into two groups at the median level, patients with higher CyD1/BCL1 expression had shorter survival (P = 0.03). This remained true when applied only to the previously untreated patients (P=0.05). Despite the relatively low expression, the CyD1/BCL1 levels in univariate analysis were as good or better predictors of survival than Binet (P = 0.03) or Rai (P = 0.05) staging. Furthermore, CyD1/BCL1 levels correlated with serum beta2-microglobulin (P = 0.001), white blood cell count (P = 0.004) and hemoglobin levels at the time of collection (P = 0.0003) but not with lymphocyte count, platelet count or age. The data demonstrate that CyD1/BCL1 is likely to play a significant role in the biology of CLL and can be used as a prognostic indicator. Further studies to clarify the role of CyD1 in the biology of CLL and its value as a prognostic indicator at the time of diagnosis are encouraged.

Topics: Adult; Aged; Aged, 80 and over; Blotting, Western; Cyclin D1; Female; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Monocytes; Prognosis; Radioimmunoassay; Survival Rate

Topics: Cyclin D1; Cyclin D2; Cyclin D3; Cyclins; Gene Expression Regulation, Leukemic; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Hairy Cell; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Lymphoproliferative Disorders; Multiple Myeloma; Neoplasm Proteins; RNA, Messenger; RNA, Neoplasm; Splenic Neoplasms

Immunophenotypic analysis is critical in categorizing small B-cell neoplasms; however, many recommended antibody panels have required fresh or frozen tissue. Many paraffin-reactive antibodies are now available but have been studied mostly in isolation. Therefore, the utility of a panel of paraffin-reactive antibodies in differentiating small B-cell neoplasms was investigated. Paraffin-embedded sections of small lymphocytic lymphoma/B-chronic lymphocytic leukemia (SLL/B-CLL; 12), mantle cell (MCL; 15), follicular (FL; 11), and marginal zone B-cell (MZL; eight) lymphomas were stained with CD20/L26, CD3, CD43/DF-T1 or Leu22, CD5/4C7, CD23/BU38, cyclin D1/H295, and CD10/56C6 antibodies. For select antibodies, results were compared to flow cytometric data (FC). Formalin and B5 fixation were also compared. Seven of 11 SLL/B-CLL were CD43+ CD5+ CD23+ cyclin D1- CD10-; seven of 11 MCL were CD43+ CD5+ CD23- cyclin D1+ CD10-; nine of 10 FL were CD43- CD5- CD23- cyclin D1- CD10+; and five of six MZL were CD43+ CD5- CD23- cyclin D1- CD10-. CD5, CD23, and CD10 stains showed sensitivities of 81, 88, and 100%, respectively, compared to FC. With B5 fixation, cyclin D1 was more often negative and CD5 more often equivocal. A panel of paraffin-reactive antibodies aids in classification of small B-cell neoplasms, although a small number of cases have indeterminate phenotypes and MZL have no defining features. CD5 separates most SLL/B-CLL and MCL from FL and MZL. CD23 separates SLL/B-CLL from most MCL, but cyclin D1 is most important for identifying MCL. CD10 positivity distinguishes most FL from other small B-cell lymphoid neoplasms.

Topics: Antigens, CD; Cyclin D1; Diagnosis, Differential; Humans; Immunohistochemistry; Immunophenotyping; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Lymphoma, Follicular; Lymphoma, Mantle-Cell; Paraffin Embedding

The cyclin-D1/PRAD1 oncogene, a key regulator of the G1-phase progression of the cell cycle, has been identified as the long-sought BCL-1 oncogene in B-cell malignancies with t(11;14)(q13;q32) translocation. A novel alternative spliced cyclin-D1 transcript, called transcript[b], has been identified. The level of the variant transcript[b] was lower than that of the originally reported cyclin-D1 transcript, called transcript[a], in several human non-lymphoid cancer cell lines but the endogenous cellular expression of transcript[b] products has not yet been determined. Northern-blot analysis and reverse-transcription-polymerase-chain-reaction (RT-PCR) analysis revealed that transcript[b] mRNA is well expressed in B-lymphoid cell lines with t(11;14)(q13;q32) translocation and at much lower or undetectable levels in other cells. Western-blot analysis using a human cyclin-D1-specific monoclonal antibody, which can recognize and distinguish the products of transcripts [a] and [b], strongly suggested that the transcript [b] protein is indeed expressed in these B-cell lines. The present study provides identification of the endogenous cellular expression of the cyclin-D1-transcript[b] protein and strongly suggests that this alternative form of cyclin D1 may play a significant role in the molecular pathogenesis of B-lymphoid malignancies with t(11;14)(q13;q32) translocation.

Topics: Alternative Splicing; Animals; Breast Neoplasms; Burkitt Lymphoma; Chromosome Mapping; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Female; Humans; Jurkat Cells; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Mice; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Translocation, Genetic; Tumor Cells, Cultured

Cyclin D1 participates in cell-cycle control, in the progression through the G(1) phase and in the transition from the G(1) to the S phase. The CCND1 locus, located in 11q13, is amplified and cyclin-D1 protein is over-expressed in a wide range of human solid tumors. In some B-lymphoid malignancies, the t(11;14)(q13;q32) translocation joins the Ig heavy-chain locus to the CCND1 locus and leads to cyclin-D1 over-expression. In this study, a series of 127 patients presenting a B-chronic lymphoproliferative disorder (B-CLPD) was analyzed using a competitive RT-PCR designed to detect cyclin-D1-mRNA over-expression. Cyclin-D1 mRNA was expressed in patients with mantle-cell lymphoma (MCL; 10/10), hairy-cell leukemia (HCL; 3/5), B-chronic lymphoid leukemia (B-CLL; 4/111) and B large-cell lymphoma (BLCL; 1/1). Densitometric analysis of RT-PCR products and Western-blot autoradiograms, in addition to cytogenetic data, indicated that activation of the cyclin-D1 gene occurred independently of the t(11;14)(q13;q32) translocation in patients with HCL. Indeed, a normal-sized protein of 36 kDa exhibiting a level incompatible with gene activation by a translocation mechanism was detected in lymphoid cells with a normal karyotype. Moreover, we found a discrepancy between cyclin-D1 mRNA and protein levels in MCL and B-CLL, which suggested that some regulatory mechanisms acting at a post-transcriptional level persist in tumor cells.

Topics: Adult; Aged; Aged, 80 and over; Blotting, Western; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Karyotyping; Leukemia, Hairy Cell; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Lymphoma, Large B-Cell, Diffuse; Male; Middle Aged; Protein Processing, Post-Translational; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Transcriptional Activation

t(11;14)(q13;q32) observed in B-cell malignancies is associated with cyclin D1 (bcl-1, PRAD1, CCND1) overexpression. We devised a simple competitive reverse transcription-polymerase chain reaction (RT-PCR) assay for rapid detection of cyclin D1 overexpression. Sharing a single upstream primer derived from a homologous sequence in cyclins D1, D2 and D3, each PCR product serves as a competitor and cyclin D1 overexpression is determined by comparing the intensities of the three amplified products. We analyzed cyclin D1 in clinical specimens from 104 patients with lymphoid malignancies. Cyclin D1 overexpression was evident in 13 of 104 (7/72 non-Hodgkin's lymphomas, 0/6 adult T-cell lymphoma/leukemias, 0/4 Hodgkin's diseases, 0/11 acute lymphoblastic leukemias, 3/4 multiple myelomas, 1/2 Waldenström's macroglobulinemias, 1/2 prolymphocytic leukemias and 1/3 chronic lymphocytic leukemias). Among 72 patients for whom cytogenetic studies had been done, all 7 patients with t(11;14) were positive. The relative expression levels of D-type cyclins altered dramatically in the presence of t(11;14). Thus, this RT-PCR assay can identify tumors with cyclin D1 overexpression. Cyclin D1 overexpression was frequent in extranodal specimens (11 out of 32 vs. 2 of 72 lymph nodes) and was restricted to specific types of lymphoid malignancies, as observed using other methods. This reliable assay should be suitable to provide clinical guidance for the diagnosis and management of lymphoid malignancies, especially in the case of extranodal involvement.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Bone Marrow; Cyclin D1; Female; Hodgkin Disease; Humans; Leukemia-Lymphoma, Adult T-Cell; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Non-Hodgkin; Lymphoproliferative Disorders; Male; Middle Aged; Multiple Myeloma; Polymerase Chain Reaction; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Transcription, Genetic; Waldenstrom Macroglobulinemia

To evaluate the usefulness of polymerase chain reaction analysis of translocations involving the bcl-1 and bcl-2 genes in variants of CD5-positive B-cell lymphomas, we analyzed four cases classified as the paraimmunoblastic variant of small lymphocytic lymphoma. This neoplasm, originally identified as an aggressive, diffuse, B-lineage lymphoma related to small lymphocytic lymphoma, can be confused with variants of mantle cell lymphoma (an immunophenotypically and morphologically similar neoplasm). No translocations involving bcl-2 and the immunoglobulin heavy chain gene were identified; two cases had translocations involving the bcl-1 and the immunoglobulin heavy chain genes. The frequency of finding this translocation suggests that these categories of neoplasms might be extremely difficult to distinguish or that a closer relationship between these neoplasms exists than was initially proposed.

Topics: Aged; Bone Marrow; Cyclin D1; Humans; Immunoglobulin Heavy Chains; Immunophenotyping; Leukemia, Lymphocytic, Chronic, B-Cell; Lymph Nodes; Male; Middle Aged; Polymerase Chain Reaction; Proto-Oncogene Proteins c-bcl-2; Translocation, Genetic

The cyclin DI/PRAD1 oncogene, a key regulator of the G1 phase of the cell cycle, has been incriminated in the pathogenesis of human neoplasia. Cyclin D1 was also demonstrated to be identical to the long-sought bcl-1 oncogene in B-cell malignancies with the t(11;14)(q13;q32) translocation. We report here a small deletion in the 3'-untranslated portion of the cyclin D1 gene in leukemia cells of a patient diagnosed with B-chronic lymphocytic leukemia (CLL), associated with overexpression of the corresponding cyclin D1 mRNA. During a Northern blot survey of B-cell malignancies, we identified a patient whose CLL cells showed a marked increase in 1.5-1.6 kb cyclin D1 mRNA species. Subsequent Southern blot analysis showed that genomic DNA from the patient's cells contained an extra band in the EcoRI digest, suggesting that one allele of the cyclin D1 gene may be altered. Polymerase chain reaction (PCR) analysis of the genomic DNA and direct DNA sequencing clearly disclosed that one allele of the cyclin D1 gene was deleted in the 3'-untranslated region, which would contribute to an increased stability of its mRNA. Reverse transcription-polymerase chain reaction (RT-PCR) analysis and direct DNA sequencing revealed that the cyclin D1 mRNA was deleted at the corresponding region. This finding provides further evidence for a critical role of cyclin D1 in the pathogenesis of B-cell malignancies and highlights a novel mechanism, a small deletion in the 3'-untranslated region, responsible for deregulation of the cyclin D1 gene in oncogenesis.

Topics: Base Sequence; Cyclin D1; Gene Deletion; Genes, bcl-1; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Molecular Sequence Data; RNA, Messenger

We report here the molecular study of a t(11;19)(q13;p13) translocation observed in a case of B-cell chronic lymphocytic leukemia. This translocation leads to the juxtaposition of the CCND1 gene on chromosome 11 to a new transcriptional unit on chromosome 19. The cDNA of this new evolutionarily conserved gene (named FLRG for Follistatin-Related Gene) codes for a secreted glycoprotein of the follistatin-module-protein family. FLRG is expressed in a wide range of human and murine adult tissues and its expression seems to be tightly regulated during murine embryogenesis. Its transcripts could not be detected in hematopoietic cells from all lineages and in particular in cells from lymphoid B and T lineage except in the t(11;19)-carrying leukemia described here. A great variability of expression is observed among the other tumoral cell lines analysed. Besides the t(11;19)-carrying leukemia described in this work, structural rearrangements of the FLRG locus have been found in a non-Hodgkin lymphoma, suggesting that it may play a role in leukemogenesis.

Topics: Amino Acid Sequence; Animals; Base Sequence; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 19; COS Cells; Cyclin D1; DNA, Complementary; Follistatin; Follistatin-Related Proteins; Glycoproteins; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Molecular Sequence Data; Sequence Homology, Amino Acid; Translocation, Genetic

The chromosomal translocation t(11;14)(q13;q32) fuses the IGH and CCND1 genes and leads to cyclin D1 overexpression. This genetic abnormality is the hallmark of mantle cell lymphoma (MCL), but is also found in some cases of atypical chronic lymphocytic leukemia (CLL), characterized by a poor outcome. For an unequivocal assessment of this specific chromosomal rearrangement on interphase cells, we developed a set of probes for fluorescence in situ hybridization (FISH). Northern blotting was performed for analysis of the cyclin D1 expression in 18 patients. Thirty-eight patients, with either a typical MCL leukemic phase (17 patients) or atypical CLL with an MCL-type immunophenotype, i.e., CD19-, CD5+, CD23-/low, CD79b/sIgM(D)++, and FMC7+ (21 patients), were analyzed by dual-color interphase FISH. We selected an IGH-specific BAC probe (covering the JH and first constant regions) and a commercially available CCND1 probe. An IGH-CCND1 fusion was detected in 28 of the 38 patients (17 typical MCL and 11 cases with CLL). Cyclin D1 was not overexpressed in two patients with typical MCL and an IGH-CCND1 fusion. In view of the poor prognosis associated with MCL and t(11;14)-positive CLL, we conclude that this set of probes is a valuable and reliable tool for a rapid diagnosis of these entities.

Topics: Adult; Aged; Aged, 80 and over; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; DNA Probes; Female; Gene Rearrangement, B-Lymphocyte, Heavy Chain; Humans; Immunoglobulin Heavy Chains; Immunophenotyping; In Situ Hybridization, Fluorescence; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Non-Hodgkin; Male; Middle Aged; Polymerase Chain Reaction; Reproducibility of Results; Translocation, Genetic

We describe a case of low-grade B-cell neoplasm with features overlapping between B-chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). The patient presented with a 10-year history of stable CLL without any treatment. The peripheral-blood picture was consistent with atypical mixed CLL (French-American-British criteria), whereas the lymph-node histology was more consistent with MCL. Neoplastic cells were strongly positive for surface immunoglobulin M, kappa, CD5, CD20, CD23, and cyclin D1. Expression of CD11c was weak. Translocation (11;14) and der(10)t(10;?)(p11;?) were the primary cytogenetic changes observed in both peripheral blood (47%) and lymph node (7%). Trisomy 12 was absent. Deletion 6q21 and rearrangements involving 1p/q, consistently associated with progression in lymphomas, also were noted in the peripheral blood but were nonclonal. The present case and similar cases with features overlapping between CLL and MCL most likely represent hybrids. In cases with features of typical CLL, t(11;14) is probably associated with gradual progression and may precede clinical and histologic transformation.

Topics: Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Disease Progression; Humans; Karyotyping; Leukemia, Lymphocytic, Chronic, B-Cell; Lymph Nodes; Lymphocytes, Tumor-Infiltrating; Lymphoma, B-Cell; Male; Middle Aged; Receptors, IgE; Translocation, Genetic

Diagnosis of small B-cell lymphomas is sometimes difficult without fresh tissue for flow cytometry (FC) or immunohistochemistry (IHC). Therefore, we examined the usefulness of a paraffin section IHC panel consisting of antibodies to CD5, CD10, CD20, CD23, CD43, and cyclin D1. We tested 55 formalin-fixed small B-cell lymphomas, including 16 small lymphocytic lymphomas (SLLs), 10 mantle cell lymphomas (MCLs), 25 follicle center lymphomas (FCLs), and 4 mantle zone lymphomas (MZLs). Seventeen cases had B5-fixed sections that were stained in the same manner. The findings were correlated with FC immunophenotyping when available. All of the SLLs and 90% of the MCLs expressed CD5 by IHC, with occasional weak expression in some MCLs. All of the FCLs and MZLs lacked CD5 expression. These results were comparable to those obtained by FC. CD43 expression was seen in 100% of the SLLs, 90% of the MCLs, and 75% of the MZLs. CD23 expression was seen in 94% of the SLL; of these, 100% also showed expression of CD23 by FC. Cyclin D1 was detected in all of the MCLs by IHC but also in 3 of the 16 SLLs. CD23 was absent in all of the MCLs. CD10 expression was present in 21 (95%) of 22 FCLs. All of the 17 cases fixed in B5 showed a decreased immunoreactivity for CD5 in the neoplastic cells. In contrast, CD10 immunoreactivity was judged better in B5-fixed sections. We concluded, therefore, that anti-CD5 and -CD10 were useful tools in the differential diagnosis of B-cell lymphomas of small lymphocytes and that a paraffin-section IHC panel consisting of antibodies to CD5, CD10, CD20, CD23, CD43, and cyclin D1 was a useful ancillary technique that compared favorably with FC.

Topics: Antigens, CD; Antigens, CD20; CD5 Antigens; Cyclin D1; Diagnosis, Differential; Fixatives; Formaldehyde; Humans; Immunohistochemistry; Leukemia, Lymphocytic, Chronic, B-Cell; Leukosialin; Lymphoma, B-Cell; Lymphoma, Follicular; Lymphoma, Non-Hodgkin; Neprilysin; Paraffin Embedding; Receptors, IgE; Sialoglycoproteins

Conventional chromosome analysis (CCA) and fluorescent in situ hybridization (FISH) studies, using a 390-kb yeast artificial chromosome probe spanning the area of multiple breakpoints of the BCL1 locus at 11q13, were performed on 57 patients fulfilling the French-American-British criteria for the diagnosis of atypical B-cell chronic lymphocytic leukemia (CLL). To better define the incidence of 13q deletions and trisomy 12, FISH analysis was also performed using a cosmid probe that recognized a DNA sequence between the Rb gene and the D13S25 locus at band 13q14 and a chromosome 12-specific pericentromeric probe. All patients were characterized by cytoimmunological and hematological studies. Fourteen cases displayed three fluorescent signals in 41-98% interphase cells when hybridized to the BCL1 yeast artificial chromosome probe, documenting the presence of BCL1 translocation (BCL1-positive cases). The presence of t(11;14)(q13;q32) was ascertained in 12 cases using CCA and by dual color interphase FISH using the BCLI probe and a 14q telomere probe in 2 karyotypically normal cases. The remaining 43 cases had two signals in more than 95% interphase cells (BCL1-negative) and did not have the t(11;14) at CCA. Although 13q14 deletions were seen by means of CCA in only 5 of 14 BCL1-positive cases, hemizygous or homozygous deletions at band 13q14 were detected by FISH in 11 of 14 BCL1-positive cases, as compared with 17 of 43 BCL1-negative cases (P = 0.01). A subclone with trisomy 12 in addition to BCL1 translocation and del(13q14) was present in four BCL1-positive cases. We arrived at the following conclusions: (a) FISH with this BCL1 YAC probe is an efficient method for the detection of the t(11;14) and of the corresponding involvement of the BCL1 locus in this lymphoproliferative disorder; (b) the majority of BCL1-positive atypical CLLs by French-American-British criteria may carry 13q14 deletions; (c) the recognition of this cytogenetic subset of atypical CLL, sharing some immunological and cytogenetic features with mantle cell lymphoma, may be important, because these patients usually present isolated peripheral blood and marrow lymphocytosis, with or without mild to moderate spleen involvement, and may require early cytotoxic treatment.

Topics: Chromosomes, Artificial, Yeast; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Female; Humans; In Situ Hybridization, Fluorescence; Karyotyping; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Proto-Oncogene Proteins; Sequence Deletion; Translocation, Genetic; Trisomy

The distinction between mantle cell lymphoma (MCL) and other low-grade B-cell neoplasms is important because MCL has a more aggressive clinical course. In bone marrow biopsy specimens, this distinction can be especially difficult. We examined 70 bone marrow biopsy specimens involved by various B-cell lymphoid neoplasms to assess the utility of cyclin D1 immunostaining in distinguishing MCL from other B-cell lymphoproliferative disorders. We used a cocktail of two monoclonal anti-cyclin D1 antibodies and a heat- and sonication-induced epitope retrieval procedure. The neoplasms assessed included MCL (32 cases), small lymphocytic lymphoma/chronic lymphocytic leukemia (18 cases), follicular lymphoma (11 cases), hairy cell leukemia (5 cases), splenic marginal zone lymphoma (2 cases), and small lymphocytic lymphoma with plasmacytoid differentiation (2 cases). The diagnosis of MCL in bone marrow was confirmed by review of the original diagnostic biopsy specimens along with additional data, such as immunophenotypic or molecular studies. Most MCL (23/32; 72%) cases expressed cyclin D1 protein. In contrast, one case of small lymphocytic lymphoma/chronic lymphocytic leukemia (1/18; 6%) and one case of hairy cell leukemia (1/5; 20%) expressed cyclin D1 protein. These findings demonstrate that immunostaining for cyclin D1 protein expression is useful in distinguishing MCL from other B-cell lymphoid neoplasms in the bone marrow.

Topics: Antibodies, Monoclonal; Base Sequence; Bone Marrow; Bone Marrow Neoplasms; Cyclin D1; Cyclins; Diagnosis, Differential; DNA Primers; DNA, Neoplasm; Humans; Immunohistochemistry; Immunophenotyping; Leukemia, Hairy Cell; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Follicular; Lymphoma, Non-Hodgkin; Oncogene Proteins; Pathology, Clinical; Polymerase Chain Reaction

The cell cycle regulatory protein cyclin D1 is essential for G1-S phase transition in several epithelial and mesenchymal tissues but is apparently not essential in normal mature B cells. An overexpression of cyclin D1 is induced by the chromosomal translocation t(11;14)(q13;q32), which characterizes non-Hodgkin's lymphomas (NHLs) of mantle cell type. We studied 26 cases of mantle cell lymphoma (MCL) for the expression of cyclins D1 and D3. A total of 23 lymphomas showed a nuclear staining for cyclin D1, whereas reactive B cells of residual germinal centers were constantly negative. When compared with cyclin D3, an inverse staining pattern emerged. Whereas the B cells of residual germinal centers reacted strongly positive for cyclin D3, there was low or missing expression of cyclin D3 in MCL cells. In other B-cell lymphomas (n = 55), including chronic lymphocytic leukemia, low-grade lymphomas of mucosa-associated lymphatic tissue, follicular lymphomas, and diffuse large B-cell lymphomas, no cyclin D1 expression could be detected and 89% of these cases displayed cyclin D3 positivity. Lymphoma cell lines harboring the t(11;14) showed cyclin D1 protein but no or very low levels of cyclin D3; three other B-cell lines, a T-cell line, and peripheral blood lymphocytes strongly expressed cyclin D3 and reacted negatively for cyclin D1. We conclude that the chromosomal translocation t(11;14) leads to an abnormal protein expression of cyclin D1 in the tumor cells of MCL and induces a consecutive downregulation of cyclin D3. In contrast to other B-NHLs, cyclin D1 and D3 expression in MCL is not related to the growth fraction.

Topics: Cell Division; Cyclin D1; Cyclin D3; Cyclins; Down-Regulation; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell, Marginal Zone; Lymphoma, Follicular; Lymphoma, Non-Hodgkin; Translocation, Genetic; Tumor Cells, Cultured

Centrocytic/mantle cell lymphoma (CC/MCL) is a morphologically defined B-cell non-Hodgkin's lymphoma characterized by a distinctive immunophenotype, BCL1/cyclin D1 (PRAD1) gene rearrangements, and, most recently, by overexpression of cyclin D1. Even using multiple breakpoint probes for BCL1 (MTC, p94PS) and cyclin D1, however, only approximately 70% of CC/MCL have a rearrangement consistent with a t(11;14) (q13;q32). To determine whether the type of molecular translocation affects the degree of cyclin D1 expression and to evaluate lymphomas diagnosed as CC/MCL but lacking molecular evidence of a BCL1 or cyclin D1 translocation, 16 CC/MCL and four cases of small lymphocytic lymphoma/B-CL1 (SLL/B-CLL) were stained using an anti-cyclin D1 antibody. All cases with a cyclin D1 translocation detected by Southern blotting techniques as well as four of the five CC/MCL without a documentable translocation showed nuclear cyclin D1 protein expression. There was no apparent correlation between staining intensity and the precise site or presence of a detectable translocation. Cases with a mantle zone growth pattern showed infiltration of the cyclin D1 positive cells into reactive follicular centers. None of the four SLL/B-CLL showed cyclin D1 expression. These findings show overexpression of the cyclin D1 protein in virtually all CC/MCL independent of the type or presence of a documentable BCL1 or cyclin D1 molecular rearrangement. The mechanism for cyclin D1 overexpression in the cases without a documentable rearrangement and the relationship of cyclin D1 overexpression to the pathogenesis of mantle cell neoplasia remain uncertain.

Topics: Cyclin D1; Cyclins; Gene Rearrangement; Humans; Immunohistochemistry; Immunophenotyping; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Non-Hodgkin; Oncogene Proteins; Staining and Labeling

The putative oncogenes BCL-1, BCL-2, and BCL-3 are commonly rearranged by translocations to the immunoglobulin genes in B-cell malignancies. However, Southern blotting rarely detected their involvement in chronic lymphocytic leukemia (CLL). This discrepancy could stem from some unique features of the oncogenesis of CLL or be due to shortcomings of Southern blotting. We have therefore evaluated the role of fluorescence in situ hybridization (FISH) in the detection of these oncogenes in CLL. Twenty consecutive CLL patients were studied by FISH for the detection of BCL-1, BCL-2, or BCL-3 rearrangement and for the presence of trisomy 12. Selected patients were also evaluated by classical cytogenetic techniques and by Southern blot analysis. Juxtaposition of JH and BCL-1 was demonstrated in 10 (50%), BCL-2 in three (15%), and BCL-3 in four (20%) of the patients. Trisomy 12 was detected by FISH in 11 (55%) patients. The coexistence of trisomy 12 and translocation of the BCL-1 oncogene was common. Three of the patients had chromosomal aberrations compatible with those detected by FISH. In contrast, in none of the five patients selected by their positive FISH findings was a rearrangement demonstrated by Southern blotting. We conclude that FISH is a sensitive method for the detection of oncogene involvement in CLL. Mainly BCL-1, but also BCL-2 and BCL-3, are commonly translocated to the immunoglobulin heavy chain locus on chromosome 14. These translocations are often associated with trisomy 12. These findings indicate that the BCL oncogenes are commonly involved in CLL and lend support to the multi-hit theory of cancer development.

Topics: Aged; Aged, 80 and over; B-Cell Lymphoma 3 Protein; Chromosome Aberrations; Chromosome Disorders; Cyclin D1; Female; Humans; In Situ Hybridization, Fluorescence; Karyotyping; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Oncogenes; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Transcription Factors

Mantle cell (centrocytic) lymphoma (MCL) and occasional cases of B-cell small lymphocytic lymphoma/chronic lymphocytic leukemia (B-SLL/CLL) show a characteristic translocation, t(11:14)(q13;q32) involving rearrangement of the Bcl-1 region. Recently it was shown that the key Bcl-1 region oncogene is cyclin D1/PRAD1; cyclin D1 mRNA was shown to be overexpressed in cases of MCL. We examined cyclin D1 protein expression in low-grade B-cell lymphomas and reactive lymphoid hyperplasias using polyclonal and monoclonal antibodies to cyclin D1 protein. Definite nuclear staining was seen in 15 of 15 MCLs, 1 of 7 B-SLL/CLLs, 0 of 7 reactive hyperplasias, 0 of 10 follicular lymphomas, and 0 of 4 lymphomas of mucosa-associated lymphoid tissue using immunoperoxidase stains on paraffin-embedded sections. Best results were obtained with the affinity-purified polyclonal antibody on microwave-treated, formalin-fixed, paraffin-embedded tissue. MCLs showed diffuse nuclear staining, whereas the one positive B-SLL/CLL showed dot-like or globular nuclear staining. Nuclear cyclin D1 protein can be detected in all cases of MCL and in rare cases of B-SLL/CLL using an immunohistochemical technique on formalin-fixed, paraffin-embedded tissue, and it does not appear to be detectable in reactive hyperplasias and other low-grade B-cell lymphomas. This protein may be useful in subclassification of low-grade B-cell lymphomas.

Topics: Blotting, Western; Cyclin D1; Cyclins; Humans; Hyperplasia; Immunohistochemistry; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Lymphoma, Follicular; Lymphoma, Non-Hodgkin; Neoplasms, Multiple Primary; Oncogene Proteins; Staining and Labeling

Employing Northern blot analysis and the polymerase chain reaction, we investigated PRAD1 gene overexpression in the tumour tissues of 58 patients with B-cell lymphoma. These findings were then examined in relation to the patients' clinical and immunohistological characteristics. The over-expression of this gene was detected in 6/8 patients with mantle cell lymphoma (MCL) and in only 1/50 other lymphomas, indicating its close association with MCL. The patients with MCL had common clinical findings of advanced disease with generalized lymphadenopathy on admission, and they had a CD5+CD10-IgD+ phenotype. The patients with chronic lymphocytic leukaemia (CLL) also showed findings indicating a distinctive disease entity: a CD5+CD10-IgD+ phenotype and lack of PRAD1 over-expression. In contrast, most patients with diffuse low-grade lymphoma other than MCL and CLL had localized extranodal disease, expressed a CD5-CD10-IgD- phenotype, and lacked PRAD1 over-expression. These findings suggest that extranodal low-grade lymphomas differ from nodal MCL and are not part of the spectrum of CLL.

Topics: Base Sequence; Blotting, Northern; Cyclin D1; Cyclins; Gene Expression; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lung Neoplasms; Lymphoma, B-Cell; Lymphoma, Non-Hodgkin; Molecular Sequence Data; Oncogene Proteins; Polymerase Chain Reaction

The t(11;14)(q13;q32) translocation and its molecular counterpart bcl-1 rearrangement are frequently associated with mantle cell lymphomas (MCLs) and only occasionally with other variants of B-cell lymphoid malignancies. This translocation seems to activate the expression of PRAD-1/cyclin D1 gene located downstream from the major breakpoint cluster region of this rearrangement. However, the possible overexpression of this gene in other lymphoproliferative disorders independently of bcl-1 rearrangement is unknown. We have examined the overexpression of PRAD-1 gene in a large series of 142 lymphoproliferative disorders including 20 MCLs by Northern blot analysis. Cytogenetic and/or bcl-1 rearrangement analysis with 2 probes (MTC, p94PS) were performed in 28 cases. Strong PRAD-1 overexpression was observed in 19 of the 20 MCLs including 3 gastrointestinal forms and 4 blastic variants. t(11;14) and/or bcl-1 rearrangement was detected in 6 of the 12 MCLs examined. No correlation was found between the different levels of mRNA expression and the pathologic characteristics of the lymphoma. Among chronic lymphoproliferative disorders other than MCL, only 1 atypical chronic lymphocytic leukemia (CLL) with a t(11;14) translocation and bcl-1 rearrangement and the 2 hairy cell leukemias (HCLs) analyzed showed upregulation of PRAD-1 gene. The expression in the 2 HCLs was lower than in MCL, and no bcl-1 rearrangement was observed. These findings indicate that PRAD-1 overexpression is a highly sensitive and specific molecular marker of MCL but it may also be upregulated in some B-CLLs and in HCL.

Topics: Biomarkers, Tumor; Blotting, Northern; Chronic Disease; Cyclin D1; Cyclins; Gene Expression; Gene Rearrangement; Humans; Leukemia, Hairy Cell; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma; Lymphoproliferative Disorders; Oncogene Proteins; Proto-Oncogene Proteins; RNA, Messenger; Translocation, Genetic

The 11q13 breakpoint region of t(11;14) (q13;q32), translocated to the Ig heavy chain locus at 14q32, has been designated as BCL-1 for B-cell leukemia/lymphoma-1, but the nature of the transcriptional unit has long remained unclear. Recently, the PRAD1 gene encoding cyclin D1, isolated from the 11q13 region, was proposed as a candidate BCL-1 gene on the basis of chromosome walking and concordant overexpression of PRAD1 mRNA in cell lines with t(11;14)(q13;q32). We report here molecular analysis of a variant translocation at the BCL-1 locus, t(11;22)(q13;q11), showing juxtaposition of the Ig light chain gene, Ig lambda, to the PRAD1 gene at its 3' end, resulting in overexpression of PRAD1 mRNA. Because only the PRAD1 gene is present between the Ig heavy chain and light chain gene breakpoints, an identity between BCL-1 and the PRAD1/cyclin D1 gene is strongly indicated.

Topics: Base Sequence; Chromosome Mapping; Chromosome Walking; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Chromosomes, Human, Pair 22; Cyclin D1; Cyclins; DNA Primers; Gene Expression; Genetic Variation; Humans; Immunoglobulin Heavy Chains; Immunoglobulin lambda-Chains; Immunoglobulin Light Chains; Introns; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma; Molecular Sequence Data; Oncogene Proteins; Polymerase Chain Reaction; RNA, Messenger; Translocation, Genetic

B cell chronic lymphocytic leukemia (B-CLL) represents the most frequent adult leukemia in the Western world. The molecular pathogenesis of B-CLL is largely unknown. Although initial reports on small panels of cases had suggested a role for Bcl-1 and Bcl-2 oncogene activation in B-CLL, later investigations failed to confirm these data. Among tumor suppressor genes, p53 mutations have been reported in a fraction of cases. In this study, we have attempted a conclusive definition of the involvement of dominantly acting oncogenes (Bcl-1 and Bcl-2) and tumor suppressor loci (p53, 6q-) in 100 cases of B-CLL selected for their CD5 positivity and Rai's stage (0 to IV). Rearrangements of Bcl-1 and Bcl-2 and deletions of 6q and 17p were analyzed by Southern blot using multiple probes. Mutational analysis (single strand conformation polymorphism and polymerase chain reaction direct sequencing) was used to assay p53 inactivation. No alterations of Bcl-1 or Bcl-2 were detected in the 100 cases tested. Mutations of p53 were found in 10/100 cases without any significant association with clinical stage. Deletions of 6q were present in 4/100 cases. Overall, our data indicate that: 1) contrary to previous reports, Bcl-1 and Bcl-2 rearrangements are not involved in CD5+ B-CLL pathogenesis and 2) p53 mutations are present in 10% of cases at all stages of the disease.

Topics: Blotting, Southern; Cyclin D1; DNA, Neoplasm; Genes, p53; Genes, Tumor Suppressor; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Oncogenes; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2

The markers, CD11b, CD11c, CD14, CD21, CD23, CD25, CD38, and FMC7 were correlated with morphologic and other laboratory and clinical characteristics of 127 patients with untreated CD5+ chronic lymphocytic leukemia (CLL). Only CD38 and CD21 were significantly associated with atypical CLL morphology. The integrin associated markers CD11b and CD11c were associated with lower leukocyte count (white blood cell count [WBC]) and lower Rai stage. By contrast, the activation antigen CD23 was associated with a higher WBC, higher Rai stage, younger age group, and the presence of lymphadenopathy. Therefore, we conclude that CD23 positivity may reflect a more aggressive form of CLL, and CD11b and CD11c positivity a less aggressive form. The BCL-1 gene rearrangement was present in 5 of 84 (6%) CLL cases examined and was associated with atypical morphology and surface expression of CD11b. Patients with a BCL-1 gene rearrangement may represent a CLL subset or possibly a different B-cell disease.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD; Antigens, Surface; CD11 Antigens; Cyclin D1; Female; Gene Rearrangement, B-Lymphocyte; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Proto-Oncogene Proteins; Receptors, IgE; Receptors, Interleukin-2

The PRAD1 gene identified from the chromosome band 11q13 region was previously demonstrated to be overexpressed in cell lines with t(11;14)(q13;q32) translocation and was suggested to be a candidate BCL-1 gene. We report here one case of mantle zone lymphoma with a t(11;22)(q13;q11), a variant translocation at the BCL-1 locus, having the PRAD1 overexpression. By analogy with the c-myc gene in Burkitt's lymphoma and the BCL-2 gene in follicular lymphoma, this case supports strongly the idea that the PRAD1 is the candidate BCL-1 gene.

Topics: Aged; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 22; Cyclin D1; Cyclins; Female; Gene Expression; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Oncogene Proteins; Translocation, Genetic

The t(11;14)(q13;q32) translocation and its molecular counterpart, BCL-1 rearrangement, are consistent features of intermediate lymphocytic lymphoma (ILL). Rearrangement is thought to deregulate the nearby PRAD-1/BCL-1 proto-oncogene that is a newly identified member of the cyclin family. To characterize further the association between rearrangement of chromosome 11q13 and over-expression of BCL-1. Southern blot analysis was performed in 33 cases of ILL, 5 cases of t(11;14)-associated leukemias, and 1 case of leukemia carrying a variant translocation t(11;19)(q13;q13) using three separate BCL-1 locus probes. When RNA was available, BCL-1 expression was assessed by Northern blot analysis. DNA from 19 of 33 ILL (57%) showed BCL-1 rearrangement, 16 involving the major translocation cluster (MTC) region and 3 involving a new breakpoint cluster located in the 5' flanking region of the BCL-1 gene. DNA from 3 of 6 t(11q13)-associated leukemias demonstrated a rearrangement involving the MTC. Northern blot analysis showed that BCL-1 was overexpressed in 14 of 15 ILL and in all leukemias analyzed (included the t(11;19) leukemia) relative to normal and malignant lymphoid tissues. These results constitute additional elements in favor of the role of BCL-1 in lymphoid neoplasia and allow us to speculate about its mechanisms of activation.

Topics: Aged; Chromosomes, Human, Pair 11; Cyclin D1; Female; Gene Expression; Gene Rearrangement; Humans; Leukemia, B-Cell; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Proto-Oncogene Mas; Proto-Oncogene Proteins; RNA, Messenger; RNA, Neoplasm; Translocation, Genetic

Previous studies using classical cytogenetics have demonstrated the presence of the t(11;14) (q13;q32) chromosomal translocation in some cases of lymphocytic lymphoma of intermediate differentiation (IDL), a distinct type of low grade B-cell lymphoma. This finding suggested that the bcl-1 region (located at band q13 of chromosome 11) might be involved in this neoplasm. Using a genomic probe from the major breakpoint area of the bcl-1 locus, we identified rearrangements of the bcl-1 region in 10 of 19 cases, 2 of which comigrated with a rearranged allele of the immunoglobulin heavy chain gene joining region. In contrast, bcl-1 rearrangements were not found in other types of low grade B-cell lymphoma, specifically in 36 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and 27 cases of follicular lymphoma (FL). To further assess the molecular pathology of IDL, we analyzed these cases for rearrangements of the bcl-2 proto-oncogene, which is associated primarily with follicular lymphomas. None of the 19 cases of IDL had rearrangements. Furthermore, none of the 36 cases of CLL/SLL showed bcl-2 rearrangements, whereas, as expected, 21 of 27 cases of FL had rearrangements of the bcl-2 locus. Our findings demonstrate an association between a rearranged bcl-1 region with approximately 50% of IDLs and suggest that abnormalities of this locus may be important in the pathogenesis of IDL.

Topics: Adult; Aged; Cell Transformation, Neoplastic; Chromosome Mapping; Chromosomes, Human, Pair 11; Cyclin D1; Female; Gene Rearrangement, B-Lymphocyte; Genotype; Humans; Immunophenotyping; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Lymphoma, Non-Hodgkin; Male; Middle Aged; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2