ABSTRACT
Previously, we reported that B-cell chronic lymphocytic leukemia (CLL) patients contained elevated levels of microvesicles (MVs). However, given the quiescent nature of CLL B-cells and the relative indolence of the disease, the dynamics of MV generation and their unique phenotypes are not clearly defined. In this study, we find that CLL B-cells generate MVs spontaneously and can be further induced by B-cell receptor-ligation. Most interestingly, CLL B-cells predominantly generate CD52+ MVs, but not CD19+ MVs in vitro, suggesting preferential usage of CD52 into leukemic-MVs and that the CLL plasma MV phenotypes corroborate well with the in vitro findings. Importantly, we detected increased accumulation of CD52+ MVs in previously untreated CLL patients with progressive disease. Finally, sequential studies on MVs in pre- and post-therapy CLL patients demonstrate that although the plasma CD52+ MV levels drop significantly after therapy in most and remain at low levels in some patients, a trend of increased accumulation of CD52+ MVs was detected in majority of post-therapy CLL patients (25 of 33). In total, this study emphasizes that dynamic accumulation of CD52+ MVs in plasma can be used to study CLL progression and may be a useful biomarker for patients as they progress and require therapy.
Subject(s)
Cell-Derived Microparticles/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Antigens, CD/metabolism , Antigens, CD19/metabolism , Antigens, Neoplasm/metabolism , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , B-Lymphocytes/metabolism , B-Lymphocytes/ultrastructure , Biomarkers , CD52 Antigen , Cell Line, Tumor , Cell-Derived Microparticles/ultrastructure , Disease Progression , Glycoproteins/metabolism , Humans , Immunophenotyping , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Time-to-Treatment , Treatment OutcomeABSTRACT
High-count monoclonal B-cell lymphocytosis (MBL) is an asymptomatic expansion of clonal B cells in the peripheral blood without other manifestations of chronic lymphocytic leukemia (CLL). Yearly, 1% of MBLs evolve to CLL requiring therapy; thus being critical to understand the biological events that determine which MBLs progress to intermediate/advanced CLL. In this study, we performed targeted deep sequencing on 48 high-count MBLs, 47 of them with 2-4 sequential samples analyzed, exploring the mutation status of 21 driver genes and evaluating clonal evolution. We found somatic non-synonymous mutations in 25 MBLs (52%) at the initial time point analyzed, including 12 (25%) with >1 mutated gene. In cases that subsequently progressed to CLL, mutations were detected 41 months (median) prior to progression. Excepting NOTCH1, TP53 and XPO1, which showed a lower incidence in MBL, genes were mutated with a similar prevalence to CLL, indicating the early origin of most driver mutations in the MBL/CLL continuum. MBLs with mutations at the initial time point analyzed were associated with shorter time-to-treatment (TTT). Furthermore, MBLs showing subclonal expansion of driver mutations on sequential evaluation had shorter progression time to CLL and shorter TTT. These findings support that clonal evolution has prognostic implications already at the pre-malignant MBL stage, anticipating which individuals will progress earlier to CLL.
Subject(s)
B-Lymphocytes/pathology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Lymphocytosis/diagnosis , Mutation , Adult , Aged , Aged, 80 and over , Cell Transformation, Neoplastic/pathology , Clonal Evolution , Clone Cells/pathology , Disease Progression , Female , Genomics , Humans , Lymphocyte Count , Lymphocytosis/genetics , Male , Middle Aged , Prognosis , Time-to-TreatmentSubject(s)
Biomarkers, Tumor/analysis , Leukemia, Lymphocytic, Chronic, B-Cell/mortality , Adult , Aged , Cohort Studies , Disease-Free Survival , Female , Humans , Male , Middle Aged , Multicenter Studies as Topic , Observational Studies as Topic , Prognosis , Proportional Hazards Models , Risk FactorsSubject(s)
Cell Survival , Fibroblast Growth Factors/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Proto-Oncogene Proteins/physiology , Receptor Protein-Tyrosine Kinases/physiology , Receptors, Fibroblast Growth Factor/metabolism , Cell Line, Tumor , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Phosphorylation , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Fibroblast Growth Factor/genetics , Axl Receptor Tyrosine KinaseABSTRACT
It is unknown whether individuals with monoclonal B-cell lymphocytosis (MBL) are at risk for adverse outcomes associated with chronic lymphocytic leukemia (CLL), such as the risk of non-hematologic cancer. We identified all locally residing individuals diagnosed with high-count MBL at Mayo Clinic between 1999 and 2009 and compared their rates of non-hematologic cancer with that of patients with CLL and two control cohorts: general medicine patients and patients who underwent clinical evaluation with flow cytometry but who had no hematologic malignancy. After excluding individuals with prior cancers, there were 107 high-count MBL cases, 132 CLL cases, 589 clinic controls and 482 flow cytometry controls. With 4.6 years median follow-up, 14 (13%) individuals with high-count MBL, 21 (4%) clinic controls (comparison MBL P<0.0001), 18 (4%) flow controls (comparison MBL P=0.0001) and 16 (12%) CLL patients (comparison MBL P=0.82) developed non-hematologic cancer. On multivariable Cox regression analysis, individuals with high-count MBL had higher risk of non-hematologic cancer compared with flow controls (hazard ratio (HR)=2.36; P=0.04) and borderline higher risk compared with clinic controls (HR=2.00; P=0.07). Patients with high-count MBL appear to be at increased risk for non-hematologic cancer, further reinforcing that high-count MBL has a distinct clinical phenotype despite low risk of progression to CLL.
Subject(s)
B-Lymphocytes , Lymphocytosis/complications , Neoplasms/etiology , Adolescent , Adult , Aged , Aged, 80 and over , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/complications , Male , Middle Aged , Proportional Hazards Models , RiskSubject(s)
Gene Expression Regulation, Leukemic , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , MicroRNAs/genetics , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Tumor Suppressor Protein p53/genetics , 3' Untranslated Regions , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , MicroRNAs/metabolism , Molecular Targeted Therapy , Proto-Oncogene Proteins/metabolism , RNA Interference , Receptor Protein-Tyrosine Kinases/metabolism , Tumor Suppressor Protein p53/metabolism , Axl Receptor Tyrosine KinaseABSTRACT
Array comparative genomic hybridization (aCGH) technology is commonly used to estimate genome-wide copy-number variation and to evaluate associations between copy number and disease. Although aCGH technology is well developed and there are numerous algorithms available for estimating copy number, little attention has been paid to the important issue of the statistical experimental design. Herein, we review classical statistical experimental designs and discuss their relevance to aCGH technology as well as their importance for downstream statistical analyses. Furthermore, we provide experimental design guidance for various study objectives.
Subject(s)
Comparative Genomic Hybridization/methods , DNA Copy Number Variations , Genetics, Population/methods , Binding, Competitive , Chromosomes, Human/genetics , DNA Probes/genetics , Genetic Association Studies , Genetic Diseases, Inborn/diagnosis , Genetic Diseases, Inborn/genetics , Genome, Human , Humans , Oligonucleotide Array Sequence Analysis , Reference Standards , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Although the risk of progression from monoclonal B-cell lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL) has been well characterized, it is unknown whether other common complications associated with CLL, such as increased risk of infection, occurs in individuals with MBL. We used the Mayo CLL database to identify cohorts of individuals with newly diagnosed MBL (n=154) or newly diagnosed CLL (n=174) who resided within 50 miles of Mayo Clinic. A cohort of 689 adult patients seen for a general medical examination who resided within 50 miles of Mayo clinic and who enrolled in a case-control study of non-Hodgkin lymphoma (NHL) was used as a comparison cohort. Hospitalization with infection was more common among individuals with MBL (25/154; 16.2%), and CLL (32/174; 18.4%) than controls (18/689; 2.6%). On pooled multivariable Cox proportional hazards analysis of all 1017 patients (controls, MBL and CLL), male sex (hazards ratio (HR)=2.3; P=0.002), major co-morbid health problems (HR=1.7, P=0.04), the presence of CLL (HR=3.2, P<0.001), treatment for progressive CLL (HR=2.4, P=0.001) and the presence of MBL (HR=3.0, P=0.001) were independently associated with risk of hospitalization for infection. These results suggest the risk of serious infection in clinical MBL is substantially greater than the risk of progression requiring treatment.
Subject(s)
Infections/etiology , Leukemia, Lymphocytic, Chronic, B-Cell/complications , Lymphocytosis/complications , Adolescent , Adult , Aged , Aged, 80 and over , Case-Control Studies , Comorbidity , Female , Follow-Up Studies , Hospitalization , Humans , Infections/mortality , Leukemia, Lymphocytic, Chronic, B-Cell/diagnosis , Leukemia, Lymphocytic, Chronic, B-Cell/mortality , Lymphocytosis/diagnosis , Lymphocytosis/mortality , Male , Middle Aged , Prognosis , Prospective Studies , Risk Factors , Survival Rate , Young AdultSubject(s)
Lymphoma, B-Cell/pathology , Age Factors , Aged , Female , Humans , Male , Middle Aged , Sex FactorsABSTRACT
Monoclonal B-cell lymphocytosis (MBL) is a hematologic condition wherein small B-cell clones can be detected in the blood of asymptomatic individuals. Most MBL have an immunophenotype similar to chronic lymphocytic leukemia (CLL), and 'CLL-like' MBL is a precursor to CLL. We used flow cytometry to identify MBL from unaffected members of CLL kindreds. We identified 101 MBL cases from 622 study subjects; of these, 82 individuals with MBL were further characterized. In all, 91 unique MBL clones were detected: 73 CLL-like MBL (CD5(+)CD20(dim)sIg(dim)), 11 atypical MBL (CD5(+)CD20(+)sIg(+)) and 7 CD5(neg) MBL (CD5(neg)CD20(+)sIg(neg)). Extended immunophenotypic characterization of these MBL subtypes was performed, and significant differences in cell surface expression of CD23, CD49d, CD79b and FMC-7 were observed among the groups. Markers of risk in CLL such as CD38, ZAP70 and CD49d were infrequently expressed in CLL-like MBL, but were expressed in the majority of atypical MBL. Interphase cytogenetics was performed in 35 MBL cases, and del 13q14 was most common (22/30 CLL-like MBL cases). Gene expression analysis using oligonucleotide arrays was performed on seven CLL-like MBL, and showed activation of B-cell receptor associated pathways. Our findings underscore the diversity of MBL subtypes and further clarify the relationship between MBL and other lymphoproliferative disorders.
Subject(s)
B-Lymphocytes/pathology , Biomarkers, Tumor/genetics , Gene Expression Profiling , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Lymphocytosis/pathology , Biomarkers, Tumor/metabolism , Flow Cytometry , Humans , Immunophenotyping , In Situ Hybridization, Fluorescence , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Oligonucleotide Array Sequence Analysis , Prognosis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The management of chronic lymphocytic leukemia (CLL) has historically relied on 'watchful waiting' and palliative approaches to therapy. However, the course of disease is highly variable and a substantial proportion of patients with early-stage CLL develop rapidly progressive disease requiring therapy. In recent decades, numerous clinical and biological prognostic markers that are predictive of decreased survival outcomes, disease progression and/or resistance to therapy, and that may play a role in defining the subgroups of patients with 'high-risk' CLL have been identified. At the same time, highly effective treatment modalities have become available with the advent of chemoimmunotherapy combinations and allogeneic stem cell transplantation. Thus, we are approaching an era when patients with CLL may potentially benefit from individualized risk assessments based on prognostic markers and when specific therapies may be offered to the subgroup of patients with high-risk disease. This review provides a brief overview of newer biological prognostic markers, discusses the challenges associated with identifying the subgroup of patients with high-risk CLL and further aims to provide recommendations on how prognostic markers may be used to assess high-risk subgroups in different clinical situations in CLL.
Subject(s)
Biomarkers, Tumor , Immunotherapy , Leukemia, Lymphocytic, Chronic, B-Cell/epidemiology , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Humans , Prognosis , Risk FactorsABSTRACT
Green tea or its constituents have long been touted as a health promoting substance including claims it may have cancer prevention properties. We previously reported the in vitro ability of one tea polyphenol, epigallocatechin gallate (EGCG), to induce apoptotic cell death in the leukemic B-cells from a majority of patients with chronic lymphocytic leukemia (CLL). After the publication of our findings many patients with CLL and other low grade lymphomas began using over-the-counter products containing tea polyphenols despite the absence of evidence to suggest clinical benefit, definition of possible toxicities, or information on optimal dose and schedule. We have become aware of four patients with low grade B-cell malignancies seen in our clinical practice at Mayo Clinic who began, on their own initiative, oral ingestion of EGCG containing products and subsequently appeared to have an objective clinical response. Three of these four patients met criteria for partial response (PR) by standard response criteria. Although spontaneous remission/regression is occasionally observed in individuals with low grade B-cell malignancies, such events are rare. Several patients presented here had documented steady clinical, laboratory, and/or radiographic evidence of progression immediately prior to initiation of over-the-counter green tea products and then developed objective responses shortly after self-initiating this therapy. Such anecdotes highlight the need for clinical trials of tea polyphenols to define the optimal dosing, schedule, toxicities, and clinical efficacy before widespread use can be recommended. An NCI sponsored phase I/II trial of de-caffeinated green tea extracts for patients with asymptomatic, early stage CLL opened at Mayo Clinic in August 2005.
Subject(s)
Anticarcinogenic Agents/therapeutic use , Camellia sinensis , Catechin/analogs & derivatives , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Phytotherapy , Camellia sinensis/chemistry , Catechin/therapeutic use , Clinical Trials as Topic , Disease-Free Survival , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/diagnostic imaging , Middle Aged , Neoplasm Regression, Spontaneous , Neoplasm Staging , Plant Extracts , Radiography , Remission Induction/methods , Tea/chemistryABSTRACT
CD38, a surface protein whose expression increases upon normal B-cell activation, is a marker of disease aggression in B-cell chronic lymphocytic leukemia (B-CLL). Higher percentages of CD38-expressing CLL B cells may be found in lymphoid compartments compared to peripheral blood. Therefore, it is possible that although CLL B cells are resting, CD38 may be a marker of recent cell activation prior to entry into the periphery. To address this hypothesis, we examined the association of CD38 expression with other activation antigens identified in gene expression profiling experiments and include CD18, CD49d, CD20, and subunit 5 of the anaphase-promoting complex/cyclosome. We found that all these markers were more highly expressed in leukemic B cells from CD38-positive CLL patients. Lastly, because interferon is known to modulate CD38 expression, we used IFN-alpha to test the ability of CLL B cells to increase CD38 expression in vitro. Interestingly, IFN stimulation only modulated CD38 expression in CLL B cells that already expressed CD38. Taken together, these data suggest that CD38 is a marker of a more recently activated CLL B cell. This in turn may explain the biological and clinical differences between CD38-positive type B-CLL and CD38-negative type B-CLL.
Subject(s)
ADP-ribosyl Cyclase 1/genetics , Interferons/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Lymphocyte Activation/drug effects , ADP-ribosyl Cyclase 1/analysis , Adult , Aged , Aged, 80 and over , Antigens, CD/analysis , Biomarkers/analysis , Female , Flow Cytometry , Gene Expression Profiling , Gene Expression Regulation , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Male , Middle Aged , PrognosisABSTRACT
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.
Subject(s)
Genes, Immunoglobulin , Immunoglobulin Heavy Chains/genetics , Interphase , Leukemia, Lymphocytic, Chronic, B-Cell/diagnosis , Oligonucleotide Probes , Translocation, Genetic , B-Cell Lymphoma 3 Protein , Cyclin D1/genetics , Diagnosis, Differential , Flow Cytometry , Genes, bcl-2 , Humans , Immunophenotyping , In Situ Hybridization, Fluorescence , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Lymphoma, B-Cell/diagnosis , Lymphoma, B-Cell/genetics , Lymphoma, Mantle-Cell/diagnosis , Lymphoma, Mantle-Cell/genetics , Proto-Oncogene Proteins/genetics , Transcription FactorsABSTRACT
The Syk family tyrosine kinase ZAP-70 is essential for normal T-cell development and signaling. Recently, leukemic cells from some patients with B-cell chronic lymphocytic leukemia (B-CLL) were shown to express ZAP-70. Owing to the prognostic value of B-CLL ZAP-70 expression, this phenotype may reflect intrinsic biological differences between the two subsets of disease. However, it remains unclear whether CLL-B cells aberrantly acquire ZAP-70 expression during the transformation process or whether ZAP-70 may be expressed under certain conditions in normal human B-lymphocytes. To discriminate between these two possibilities, we assessed ZAP-70 expression in normal human B-lymphocytes. Our data demonstrate that ZAP-70 is expressed in a subpopulation of tonsillar and splenic normal B-lymphocytes that express an activated phenotype. Furthermore, ZAP-70 expression can be induced in vitro upon stimulation of blood and tonsillar B cells. Finally, we show that phosphorylation of ZAP-70 occurs in tonsillar B cells with stimulation through the B-cell receptor. These results provide new insight into normal human B-cell biology as well as provide clues about the transformed cell in B-CLL.
Subject(s)
B-Lymphocytes/physiology , Lymphocyte Activation/physiology , Lymphocyte Subsets/physiology , Protein-Tyrosine Kinases/genetics , Cell Differentiation/immunology , Flow Cytometry , Humans , Jurkat Cells , Palatine Tonsil/cytology , Phenotype , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Spleen/cytology , Transfection , ZAP-70 Protein-Tyrosine KinaseABSTRACT
We have previously shown that chronic lymphocytic leukemia (CLL) B cells secrete vascular endothelial growth factor (VEGF) in vitro, have constitutively active VEGF receptors R1 and R2, and respond to exogenous VEGF by specifically upregulating Mcl-1 and XIAP in association with decreased cell death. We found that epigallocatechin (EGCG) decreases VEGF receptor phosphorylation and induces apoptosis in CLL B cells. The mechanism(s) by which VEGF receptor activation increases Mcl-1 and XIAP and promotes survival remains unknown. To further define the signaling pathway mediating VEGF induction of antiapoptotic proteins in CLL B-cells, we investigated downstream effects of VEGF-VEGF receptor binding on the STAT signaling pathway. We find that CLL B cells abundantly express cytoplasmic serine phosphorylated (p)-STAT-1 and p-STAT-3, VEGF-R1/2 are physically associated with p-STAT-1 and p-STAT-3, and p-STAT-3 (but not p-STAT-1) is found in the CLL nucleus. VEGF receptor ligation selectively induces activation and perinuclear translocation of STAT 3 through receptor-mediated endocytosis. The inhibition of VEGF receptor activation with either tyrosine kinase inhibitors or VEGF neutralizing antibodies inhibit VEGF receptor phosphorylation, decrease p-STAT-3 (serine 727), Mcl-1, and induces cell death in CLL B cells. Thus, a VEGF-VEGF receptor pathway in CLL B cells can be linked to activation of STAT proteins that are able to enhance their apoptotic resistance.