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1.  Targeting ROR1 Inhibits Epithelial-Mesenchymal Transition and Metastasis 
Cancer research  2013;73(12):10.1158/0008-5472.CAN-12-3832.
Metastasis is responsible for 90% of cancer-related deaths. Strategies are needed that can inhibit the capacity of cancer cells to migrate across anatomic barriers and colonize distant organs. Here we show an association between metastasis and expression of a type I receptor-tyrosine-kinase-like orphan receptor, ROR1, which is expressed during embryogenesis and by various cancers, but not by normal post-partum tissues. We found that expression of ROR1 associates with the epithelial-mesenchymal transition (EMT), which occurs during embryogenesis and cancer metastasis. Breast adenocarcinomas expressing high-levels ROR1 were more likely to have gene-expression signatures associated with EMT and had higher rates of relapse and metastasis than breast adenocarcinomas expressing low-levels of ROR1. Suppressing expression of ROR1 in metastasis-prone breast-cancer cell-lines, MDA-MB-231, HS-578T, or BT549, attenuated expression of proteins associated with EMT (e.g. vimentin, SNAIL-1/2, and ZEB1), enhanced expression of E-cadherin, epithelial cytokeratins (e.g. CK-19), and tight-junction proteins (e.g. ZO-1), and impaired their migration/invasion capacity in vitro and the metastatic potential of MDA-MB-231 cells in immune-deficient mice. Conversely, transfection of MCF-7 cells to express ROR1 reduced expression of E-cadherin and CK-19, but enhanced expression of SNAIL-1/2 and vimentin. Treatment of MDA-MB-231 with a mAb specific for ROR1 induced down-modulation of vimentin, and inhibited cancer-cell migration and invasion in vitro and tumor metastasis in vivo. Collectively, this study indicates that ROR1 may regulate EMT and metastasis, and that antibodies targeting ROR1 can inhibit cancer progression and metastasis.
PMCID: PMC3832210  PMID: 23771907
ROR1; breast cancer; metastasis; monoclonal antibody; EMT
2.  Quantitative DNA Methylation Analysis Identifies a Single CpG Dinucleotide Important for ZAP-70 Expression and Predictive of Prognosis in Chronic Lymphocytic Leukemia 
Journal of Clinical Oncology  2012;30(20):2483-2491.
Increased ZAP-70 expression predicts poor prognosis in chronic lymphocytic leukemia (CLL). Current methods for accurately measuring ZAP-70 expression are problematic, preventing widespread application of these tests in clinical decision making. We therefore used comprehensive DNA methylation profiling of the ZAP-70 regulatory region to identify sites important for transcriptional control.
Patients and Methods
High-resolution quantitative DNA methylation analysis of the entire ZAP-70 gene regulatory regions was conducted on 247 samples from patients with CLL from four independent clinical studies.
Through this comprehensive analysis, we identified a small area in the 5′ regulatory region of ZAP-70 that showed large variability in methylation in CLL samples but was universally methylated in normal B cells. High correlation with mRNA and protein expression, as well as activity in promoter reporter assays, revealed that within this differentially methylated region, a single CpG dinucleotide and neighboring nucleotides are particularly important in ZAP-70 transcriptional regulation. Furthermore, by using clustering approaches, we identified a prognostic role for this site in four independent data sets of patients with CLL using time to treatment, progression-free survival, and overall survival as clinical end points.
Comprehensive quantitative DNA methylation analysis of the ZAP-70 gene in CLL identified important regions responsible for transcriptional regulation. In addition, loss of methylation at a specific single CpG dinucleotide in the ZAP-70 5′ regulatory sequence is a highly predictive and reproducible biomarker of poor prognosis in this disease. This work demonstrates the feasibility of using quantitative specific ZAP-70 methylation analysis as a relevant clinically applicable prognostic test in CLL.
PMCID: PMC3397783  PMID: 22564988
4.  Association of a MicroRNA/TP53 Feedback Circuitry With Pathogenesis and Outcome of B-Cell Chronic Lymphocytic Leukemia 
Chromosomal abnormalities (namely 13q, 17p, and 11q deletions) have prognostic implications and are recurrent in chronic lymphocytic leukemia (CLL), suggesting that they are involved in a common pathogenetic pathway; however, the molecular mechanism through which chromosomal abnormalities affect the pathogenesis and outcome of CLL is unknown.
To determine whether the microRNA miR-15a/miR-16-1 cluster (located at 13q), tumor protein p53 (TP53, located at 17p), and miR-34b/miR-34c cluster (located at 11q) are linked in a molecular pathway that explains the pathogenetic and prognostic implications (indolent vs aggressive form) of recurrent 13q, 17p, and 11q deletions in CLL.
Design, Setting, and Patients
CLL Research Consortium institutions provided blood samples from untreated patients (n=206) diagnosed with B-cell CLL between January 2000 and April 2008. All samples were evaluated for the occurrence of cytogenetic abnormalities as well as the expression levels of the miR-15a/miR-16-1 cluster, miR-34b/miR-34c cluster, TP53, and zeta-chain (TCR)–associated protein kinase 70kDa (ZAP70), a surrogate prognostic marker of CLL. The functional relationship between these genes was studied using in vitro gain- and loss-of-function experiments in celllines and primary samples and was validated in a separate cohort of primary CLL samples.
Main Outcome Measures
Cytogenetic abnormalities; expression levels of the miR-15a/miR-16-1 cluster, miR-34 family, TP53 gene, downstream effectors cyclindependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) and B-cell CLL/lymphoma 2 binding component 3 (BBC3), and ZAP70 gene; genetic interactions detected by chromatin immunoprecipitation.
In CLLs with13qdeletions the miR-15a/miR-16-1 cluster directly targetedTP53 (mean luciferase activity for miR-15a vs scrambled control, 0.68 relative light units (RLU) [95%confidence interval {CI}, 0.63–0.73]; P=.02;meanfor miR-16 vs scrambled control, 0.62RLU[95%CI, 0.59–0.65]; P=.02) and its downstream effectors. In leukemic cell lines and primary CLL cells, TP53 stimulated the transcription of miR-15/miR-16-1 as well as miR-34b/miR-34c clusters, and the miR-34b/miR-34c cluster directly targeted theZAP70 kinase(meanluciferase activity for miR-34a vs scrambled control, 0.33RLU [95%CI, 0.30–0.36]; P=.02;meanformiR-34bvsscrambledcontrol,0.31RLU [95%CI, 0.30–0.32];P=.01; and mean for miR-34c vs scrambled control, 0.35 RLU [95% CI, 0.33–0.37]; P=.02).
A microRNA/TP53 feedback circuitry is associated with CLL pathogenesis and outcome. This mechanism provides a novel pathogenetic model for the association of 13q deletions with the indolent form of CLL that involves microRNAs, TP53, and ZAP70
PMCID: PMC3690301  PMID: 21205967
5.  Comparison of Familial and Sporadic Chronic Lymphocytic Leukaemia Using High Resolution Array Comparative Genomic Hybridization 
British journal of haematology  2010;151(4):336-345.
Approximately 10% of patients with chronic lymphocytic leukaemia (CLL) have a family history of the disease or a related lymphoproliferative disorder, yet the relationship of familial CLL to genomic abnormalities has not been characterized in detail. We therefore studied 75 CLL patients, half familial and half sporadic, using high-resolution array comparative genomic hybridization (CGH), in order to better define the relationship of genomic abnormalities to familial disease and other biological prognostic factors. Our results showed that the most common high-risk deletion in CLL, deletion 11q, was significantly associated with sporadic disease. Comparison of familial to sporadic disease additionally identified a copy number variant region near the centromere on 14q, proximal to IGH@, in which gains were associated both with familial CLL, and with mutated IGHV and homozygous deletion of 13q. Homozygous deletion of 13q was also found to be associated with mutated IGHV and low expression of ZAP-70, and a significantly longer time to first treatment compared to heterozygous deletion or lack of alteration. This study is the first high resolution effort to investigate and report somatic genetic differences between familial and sporadic CLL.
PMCID: PMC3584328  PMID: 20812997
CLL; familial; deletion 11q
6.  ROR1 is expressed on hematogones (non-neoplastic human B-lymphocyte precursors) and a minority of precursor-B acute lymphoblastic leukemia 
Leukemia research  2011;35(10):1390-1394.
ROR1 is a receptor tyrosine kinase expressed during embryogenesis, on chronic lymphocytic leukemia (CLL) and in other malignancies. Hematogones (non-neoplastic B-lymphocyte precursors) express surface ROR1 at an intermediate stage of maturation that lacks CD34 or TdT. The neoplastic counterpart to hematogones is precursor-B acute lymphoblastic leukemia (B-ALL), but less than 10% of B-ALL express surface ROR1, and these ROR1+ B-ALL cases have an unusually high frequency of lacking CD34 and/or having t(1;19), a chromosomal translocation that defines a specific subtype of B-ALL.
PMCID: PMC3163753  PMID: 21813176
ROR1; Wnt; B-lymphocyte precursors; Hematogones; Acute lymphoblastic leukemia; t(1;19); Chronic lymphocytic leukemia; Flow cytometry
7.  Cyclic nucleotide phosphodiesterase 7B mRNA: an unfavorable characteristic in chronic lymphocytic leukemia 
A cost- and time-efficient means to define the prognosis of patients with chronic lymphocytic leukemia (CLL) is desirable but does not yet exist. Based on evidence that CLL cells have enhanced expression of the cyclic nucleotide phosphodiesterase isoform 7B (PDE7B), we hypothesized that PDE7B expression might provide such information. We assessed PDE7B mRNA expression using quantitative real-time PCR (QPCR) in peripheral blood mononuclear cells isolated from 85 patients and 30 normal subjects. We compared PDE7B mRNA expression with that of other disease features to determine if its expression correlates with the prognosis of patients with CLL. We found that CLL patients with PDE7B mRNA levels in the top quartile (>9-fold elevation relative to normal controls) have a several-year shorter median time-to-treatment (TTT, 36 months) compared to that of patients whose CLL cells express lower levels of PDE7B mRNA (TTT, 77 months, P=0.001). High PDE7B mRNA expression correlates with expression of Zeta-chain-associated protein kinase 70 (ZAP-70), unmutated immunoglobulin heavy chain variable (IGHV) region genes and β2 microglobulin (β2M), but use of a multivariate Cox model revealed that high PDE7B mRNA expression independently predicts a short TTT, even after adjusting for several other disease characteristics (ZAP-70 or CD38 expression, IGHV mutation status, Rai status). High expression of PDE7B is an unfavorable characteristic in CLL. Assessment of PDE7B mRNA expression thus appears to be a clinically useful biomarker to define the prognosis of patients with CLL.
PMCID: PMC3111850  PMID: 21120911
cyclic nucleotide phosphodiesterase 7B; chronic lymphocytic leukemia; prognostic factor; quantitative reverse transcriptase polymerase chain reaction (QPCR)
8.  Immnuophenotypic and Gene Expression Analysis of Monoclonal B Cell Lymphocytosis Shows Biologic Characteristics Associated With Good Prognosis CLL 
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. Ninety-one unique MBL clones were detected: 73 CLL-like MBL (CD5+CD20dimsIgdim), 11 atypical MBL (CD5+CD20+sIg+), and 7 CD5neg MBL (CD5negCD20+sIgneg). 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 7 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.
PMCID: PMC3164475  PMID: 21617698
9.  Stimulation of Chronic Lymphocytic Leukemia (CLL) Cells with CpG Oligodeoxynucleotide (ODN) Gives Consistent Karyotypic Results among Laboratories: a CLL Research Consortium (CRC)h Study 
Cancer genetics and cytogenetics  2010;203(2):134-140.
Cytogenetic abnormalities in CLL are important prognostic indicators. Historically, only interphase cytogenetics was clinically useful in CLL because traditional mitogens are not effective mitotic stimulants. Recently, CpG-oligodeoxynucleotide (ODN) stimulation has shown effectiveness in CLL. The CLL Research Consortium (CRC) tested the effectiveness and reproducibility of CpG-ODN stimulation to detect chromosomally abnormal clones by five laboratories. More clonal abnormalities were observed after culture of CLL cells with CpG-ODN than with pokeweed mitogen (PWM)+12-O-tetradecanoyl-phorobol-13-acetate (TPA). All clonal abnormalities in PWM+TPA cultures were observed in CpG-ODN cultures, whereas CpG-ODN identified some clones not found by PWM+TPA. CpG-ODN stimulation of one normal control and 12 CLL samples showed that excepting clones of del(13q) in low frequencies and one translocation, results in all five laboratories were consistent, and all abnormalities were concordant with FISH. Thus, abnormal clones in CLL are more readily detected with CpG-ODN stimulation than with traditional B-cell mitogens. After CpG-ODN stimulation, abnormalities were reproducible among cytogenetic laboratories. CpG-ODN did not appear to induce aberrations in cell culture and enhanced detection of abnormalities and complexity in CLL. Since karyotypic complexity is prognostic and is not detectable by standard FISH analyses, stimulation with CpG-ODN is useful to identify this additional prognostic factor in CLL.
PMCID: PMC3018693  PMID: 21156225
10.  Common Occurrence of Monoclonal B-cell Lymphocytosis Among Members of High-Risk CLL Families 
British journal of haematology  2010;151(2):152-158.
Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic haematological condition characterized by low absolute levels of B-cell clones with a surface immunophenotype similar to that of chronic lymphocytic leukaemia (CLL). In the general population, MBL increases with age with a prevalence of 5–9% in individuals over age 60 years. It has been reported to be higher among first-degree relatives from CLL families. We report results of multi-parameter flow cytometry among 505 first-degree relatives with no personal history of lymphoproliferative disease from 140 families having at least two cases of CLL. Seventeen percent of relatives had MBL. Age was the most important determinant where the probability for developing MBL by age 90 years was 61%. MBL clustered in certain families but clustering was independent of the number of known CLL cases in a family. As is the case with CLL, males had a significantly higher risk for MBL than did females (p=0.04). MBL patients had significantly higher mean absolute lymphocyte counts (2.4 × 109/l) and B-cell counts (0.53 × 109/l) than those with a normal B-cell immunophenotype. Our findings show that MBL occurs at a very high rate in high risk CLL families. Both the age and gender distribution of MBL are parallel to CLL, implying a shared inherited risk.
PMCID: PMC2966536  PMID: 20738309
chronic lymphocytic leukaemia; high risk families; monoclonal B-cell lymphocytosis; flow cytometry
11.  Non-codingRNA sequence variations in human chronic lymphocytic leukemia and colorectal cancer 
Carcinogenesis  2009;31(2):208-215.
Cancer is a genetic disease in which the interplay between alterations in protein-coding genes and non-coding RNAs (ncRNAs) plays a fundamental role. In recent years, the full coding component of the human genome was sequenced in various cancers, whereas such attempts related to ncRNAs are still fragmentary. We screened genomic DNAs for sequence variations in 148 microRNAs (miRNAs) and ultraconserved regions (UCRs) loci in patients with chronic lymphocytic leukemia (CLL) or colorectal cancer (CRC) by Sanger technique and further tried to elucidate the functional consequences of some of these variations. We found sequence variations in miRNAs in both sporadic and familial CLL cases, mutations of UCRs in CLLs and CRCs and, in certain instances, detected functional effects of these variations. Furthermore, by integrating our data with previously published data on miRNA sequence variations, we have created a catalog of DNA sequence variations in miRNAs/ultraconserved genes in human cancers. These findings argue that ncRNAs are targeted by both germ line and somatic mutations as well as by single-nucleotide polymorphisms with functional significance for human tumorigenesis. Sequence variations in ncRNA loci are frequent and some have functional and biological significance. Such information can be exploited to further investigate on a genome-wide scale the frequency of genetic variations in ncRNAs and their functional meaning, as well as for the development of new diagnostic and prognostic markers for leukemias and carcinomas.
PMCID: PMC2812567  PMID: 19926640
12.  Genome-wide association study of follicular lymphoma identifies a risk locus at 6p21.32 
Nature genetics  2010;42(8):661-664.
To identify susceptibility loci for non-Hodgkin lymphoma (NHL) subtypes, we conducted a three-stage genome-wide association study. We identified two variants associated with follicular lymphoma (FL) in 1,465 FL cases/6,958 controls at 6p21.32 (rs10484561, rs7755224, r2=1.0; combined p-values=1.12×10-29, 2.00×10-19), providing further support that MHC genetic variation influences FL susceptibility. Confirmatory evidence of a previously reported association was also found between chronic lymphocytic leukemia/small lymphocytic lymphoma and rs735665 (combined p-value=4.24×10-9).
PMCID: PMC2913472  PMID: 20639881
13.  Computational Identification Of CDR3 Sequence Archetypes Among Immunoglobulin Sequences in Chronic Lymphocytic Leukemia 
Leukemia research  2008;33(3):368-376.
The leukemia cells of unrelated patients with chronic lymphocytic leukemia (CLL) display a restricted repertoire of immunoglobulin (Ig) gene rearrangements with preferential usage of certain Ig gene segments. We developed a computational method to rigorously quantify biases in Ig sequence similarity in large patient databases and to identify groups of patients with unusual levels of sequence similarity. We applied our method to sequences from 1577 CLL patients through the CLL Research Consortium (CRC), and identified 67 similarity groups into which roughly 20% of all patients could be assigned. Immunoglobulin light chain class was highly correlated within all groups and light chain gene usage was similar within sets. Surprisingly, over 40% of the identified groups were composed of somatically mutated genes. This study significantly expands the evidence that antigen selection shapes the Ig repertoire in CLL.
PMCID: PMC2692898  PMID: 18640719
14.  An international standardization programme towards the application of gene expression profiling in routine leukaemia diagnostics: the Microarray Innovations in LEukemia study prephase 
British Journal of Haematology  2008;142(5):802-807.
Gene expression profiling has the potential to enhance current methods for the diagnosis of haematological malignancies. Here, we present data on 204 analyses from an international standardization programme that was conducted in 11 laboratories as a prephase to the Microarray Innovations in LEukemia (MILE) study. Each laboratory prepared two cell line samples, together with three replicate leukaemia patient lysates in two distinct stages: (i) a 5-d course of protocol training, and (ii) independent proficiency testing. Unsupervised, supervised, and r2 correlation analyses demonstrated that microarray analysis can be performed with remarkably high intra-laboratory reproducibility and with comparable quality and reliability.
PMCID: PMC2654477  PMID: 18573112
microarray; gene expression profiling; leukaemia; standardization; diagnostics
15.  Relation of Gene Expression Phenotype to Immunoglobulin Mutation Genotype in B Cell Chronic Lymphocytic Leukemia 
The Journal of Experimental Medicine  2001;194(11):1639-1648.
The most common human leukemia is B cell chronic lymphocytic leukemia (CLL), a malignancy of mature B cells with a characteristic clinical presentation but a variable clinical course. The rearranged immunoglobulin (Ig) genes of CLL cells may be either germ-line in sequence or somatically mutated. Lack of Ig mutations defined a distinctly worse prognostic group of CLL patients raising the possibility that CLL comprises two distinct diseases. Using genomic-scale gene expression profiling, we show that CLL is characterized by a common gene expression “signature,” irrespective of Ig mutational status, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Nonetheless, the expression of hundreds of other genes correlated with the Ig mutational status, including many genes that are modulated in expression during mitogenic B cell receptor signaling. These genes were used to build a CLL subtype predictor that may help in the clinical classification of patients with this disease.
PMCID: PMC2193523  PMID: 11733578
cDNA microarrays; gene expression profiling; leukemia; lymphocytic; chronic
16.  The soluble CD40 ligand sCD154 in systemic lupus erythematosus 
Journal of Clinical Investigation  1999;104(7):947-955.
We found that the plasma of patients with active systemic lupus erythematosus (SLE) could induce a human B-cell line (Ramos) to express high levels of immune accessory molecules that are commonly found on blood B cells of patients with active SLE. The ability of SLE plasma to induce such phenotypic changes could be abrogated by neutralizing antibodies specific for the CD40 ligand (CD154) but not by antibodies to TNF-α. Immunoprecipitation studies with anti-CD154 identified a 20-kDa protein in the plasma of SLE patients with active disease, but not in plasma of normal donors, indicating that such plasma contained soluble CD154 (sCD154). Using a quantitative ELISA method, we found that the plasma of patients with active disease had levels of sCD154 that were significantly higher than those found in plasma of normal donors. Levels of CD154 transcripts in SLE blood lymphocytes correlated with the relative concentrations of sCD154 found in SLE plasma. Furthermore, plasma levels of sCD154 correlated with the titers of anti–double-stranded DNA autoantibody and with clinical disease activity. These studies indicate that sCD154 of patients with SLE may act as a functional ligand for CD40 that is associated with SLE disease activity.
PMCID: PMC408556  PMID: 10510335
17.  Lack of Allelic Exclusion in B Cell Chronic Lymphocytic Leukemia 
The Journal of Experimental Medicine  1997;185(8):1435-1446.
We determined the immunoglobulin (Ig) VH subgroup expressed by the leukemia cells of 108 patients with B cell chronic lymphocytic leukemia (CLL). Surprisingly, we found that six samples (5%) each expressed Ig of more than one VH subgroup. Southern blot analysis demonstrated that these samples each had rearrangements involving both Ig heavy chain alleles. Nucleic acid sequence analyses of the Ig cDNA revealed each to express two functional Ig VH genes: VH3-33 and VH4-39; VH3-7 and VH4-39; VH3-23 and VH4-61; VH2-70 and VH3-30.3; or VH3-30 and VH4-b (DP67). One sample expressed three Ig VH genes: VH2-70, VH3-7, and VH4-59. Despite having more than one Ig heavy chain transcript, each sample was found to express only one functional Ig light chain. From the primary sequence, we deduced that the Ig of some of these CLL samples should react with Lc1, a monoclonal antibody (mAb) reactive with a supratypic cross-reactive idiotype present on Ig encoded by a subgroup of Ig VH4 genes (namely, VH4-39, VH4-b [DP-67], VH4-59, or VH4-61), and B6, an mAb that reacts with Ig encoded by certain Ig VH3 genes (namely, VH3-23, VH3-30, or VH3-30.3), and/or modified staphylococcal protein A (SpA), a 45-kilodalton bacterial “superantigen” that reacts with most Ig of the VH3 subgroup. Flow cytometric analyses revealed that such samples did in fact react with Lc1 and B6 and/or SpA, but not with control mAbs of irrelevant specificity. This study demonstrates that a subset of CLL patients have leukemic B cells that express more than one functional Ig heavy chain.
PMCID: PMC2196272  PMID: 9126924

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