Background MicroRNA expression profiles can be used to distinguishnormal B cells from malignant B cells in patients with chroniclymphocytic leukemia (CLL). We investigated whether microRNAprofiles are associated with known prognostic factors in CLL.
Methods We evaluated the microRNA expression profiles of 94samples of CLL cells for which the level of expression of 70-kDzeta-associated protein (ZAP-70), the mutational status of therearranged immunoglobulin heavy-chain variable-region (IgVH)gene, and the time from diagnosis to initial treatment wereknown. We also investigated the genomic sequence of 42 microRNAgenes to identify abnormalities.
Results A unique microRNA expression signature composed of 13genes (of 190 analyzed) differentiated cases of CLL with lowlevels of ZAP-70 expression from those with high levels andcases with unmutated IgVH from those with mutated IgVH. Thesame microRNA signature was also associated with the presenceor absence of disease progression. We also identified a germ-linemutation in the miR-16-1–miR-15a primary precursor, whichcaused low levels of microRNA expression in vitro and in vivoand was associated with deletion of the normal allele. Germ-lineor somatic mutations were found in 5 of 42 sequenced microRNAsin 11 of 75 patients with CLL, but no such mutations were foundin 160 subjects without cancer (P<0.001).
Conclusions A unique microRNA signature is associated with prognosticfactors and disease progression in CLL. Mutations in microRNAtranscripts are common and may have functional importance.
Chronic lymphocytic leukemia (CLL), the most common leukemiaamong adults in the Western world, arises from a malignant cloneof B cells, but little is known regarding its initiation andprogression.1 Nevertheless, several factors that can predictthe clinical course have been identified.2,3,4,5,6 Cases inwhich the leukemic cells have few or no mutations in the immunoglobulinheavy-chain variable-region (IgVH) gene or a high level of expressionof the 70-kD zeta-associated protein (ZAP-70) have an aggressivecourse, whereas cases involving mutated CLL clones or few ZAP-70cells have an indolent course.7 Genomic aberrations in CLL arealso independent predictors of disease progression and survival.8However, the molecular basis of these associations is largelyunknown.
The most frequent deletion of genomic DNA in CLL occurs in chromosome13q13.4. This deletion is evident in about 50 percent of casesand is associated with a long interval between diagnosis andthe need for treatment (the treatment-free interval).8 The 13q13.4deletion is frequently the sole abnormality in CLL and othertypes of cancers,9 suggesting a pathogenic role for the deletedgene or genes. We used positional cloning to identify two membersof a recently discovered class of small, noncoding RNAs, ormicroRNAs, miR-15a and miR-16-1, which are located in the smallestregion of the deletion at 13q13.4 and are frequently deletedor down-regulated in CLL cells.10
MicroRNAs range in size from 19 to 25 nucleotides and are typicallyexcised from a hairpin (fold-back) RNA structure of 60 to 110nucleotides (named pre-microRNA) that is transcribed from alarger primary transcript (named pri-microRNA).11 MicroRNAscan reduce the levels of many of their target transcripts aswell as the amount of protein encoded by these transcripts.12The finding that approximately 50 percent of the known humanmicroRNAs are located at cancer-associated regions of the genome13suggests that microRNAs play a role in the pathogenesis of varioushuman cancers.10,14,15,16,17,18,19 Using a microRNA microchip,20we found that CLL cells (which are CD5+ B cells) have a distinctpattern of expression of microRNA that differs from that ofnormal CD5+ B cells.21
We performed genome-wide expression profiling with the microRNAmicrochip in a large number of CLL samples from patients withavailable clinical data to investigate whether the expressionof noncoding microRNA genes is associated with factors thatpredict the clinical course of CLL. We also screened severalmicroRNAs for mutations in a panel of CLL cells.
Methods
Patients and Clinical Database
For the expression study, we investigated 94 samples of CLLcells after obtaining written informed consent from patientsreceiving care at CLL Research Consortium institutions.2,10Clinical and biologic information (sex, age at diagnosis, treatment,time between diagnosis and therapy, the level of ZAP-70 expression,and mutational status of IgVH) was available for all patients(Table 1). A second independent set of 50 samples of CLL cellsfor which the level of ZAP-70 expression was known was usedto validate the predictive power of the microRNA signature.
RNA Extraction, Northern Blotting, and MicroRNA-Microchip Experiments
RNA extraction, Northern blotting, and microRNA-microchip procedureswere performed as described in detail elsewhere.20,21 Briefly,labeled targets from 5 µg of total RNA were used for hybridizationon each microRNA microchip, which contained triplicates of 368probes, corresponding to 245 human and mouse microRNA genes.We tested 76 microRNAs on the microRNA microchip with two specificsynthetic oligomers; one identified the active 22-nucleotidepart of the molecule, and the other detected the precursor composedof 60 to 110 nucleotides.20
Statistical Analysis
Raw data were normalized and analyzed with the use of GeneSpringsoftware (version 7.2, Silicon Genetics). Expression data werecentered around a median with the use of the GeneSpring normalizationoption alone and with global-median normalization containedin the Bioconductor package (www.bioconductor.org), with nosubstantial difference in results. Statistical comparisons weremade with the use of both the GeneSpring analysis-of-variancetool and the Significance Analysis of Microarray (SAM) software(available at www-stat.stanford.edu/~tibs/SAM/index.html). MicroRNApredictors were calculated with the use of Prediction Analysisof Microarray (PAM) software (available at www-stat.stanford.edu/~tibs/PAM/index.html);the Support Vector Machine tool of GeneSpring was used for cross-validationand prediction of the test set. The Kaplan–Meier plot(survival-analysis portion of the PAM software) was used toidentify any association between microRNA expression and thetime from diagnosis to the beginning of therapy. MicroRNAs thatbest differentiated among groups of patients were identifiedat the same time. All data were submitted to the Array Expressdatabase with the use of MIAMExpress (accession numbers E-TABM-41and E-TABM-42). We validated the microarray data for four microRNAs(miR-16-1, miR-26a, miR-206, and miR-223) in 11 CLL samplesand normal CD5+ cells by means of solution hybridization detectionas described elsewhere.21 Furthermore, the expression of miR-15aand miR-16-1 in the patients with a germ-line mutation was confirmedby Northern blotting.
Analysis of ZAP-70 and Sequence Analysis of IgVH
Analysis of ZAP-70 and sequence analysis of IgVH were performedas described previously.2 Briefly, ZAP-70 expression was assessedby immunoblotting and flow cytometry, whereas the analysis ofexpressed IgVH was done by direct sequencing.
Detection of MicroRNA Mutations
The genomic region corresponding to each precursor microRNAfrom 40 samples of CLL cells and normal mononuclear cells fromthree control subjects was amplified, including at least 50bp at the 5' and 3' ends. For the microRNAs located in clustersless than 1 kb apart, the entire corresponding genomic regionwas amplified and sequenced with the use of the Applied BiosystemsDNA sequencing system (model 377, Applied Biosystems). Whena deviation from the normal sequence was found, a panel of DNAsfrom the blood of 160 control subjects was screened to identifypolymorphisms, as was an additional panel from the blood of35 patients with CLL (for a total of 75 patients with leukemia).All subjects were white, as indicated by medical records orinformation obtained during an interview with control subjects.The personal and family history of cancer was known for 46 patientswith CLL.
In Vivo Studies of the Effects of miR-16-1 Mutants
We constructed miR-16-1 and miR-15a expression vectors containingan 832-bp genomic sequence including both miR-16-1 and miR-15a,one wild-type sequence, and one containing the (CT)+7 substitution,by ligating the relevant open reading frame in a sense orientationinto a mammalian expression vector pSR-GFP-Neo (OligoEngine).All sequenced constructs were transfected in 293 cells withthe use of Lipofectamine2000 according to the manufacturer'sprotocol (Invitrogen). The expression of both constructs wasassessed by Northern blotting.
Results
MicroRNA Signature, ZAP-70 Expression, and Mutational Status of IgVH
We investigated whether the microRNA-microchip microarray couldreveal a specific molecular signature that is associated withsubgroups of CLL with different clinical courses. Using 20 percentas the cutoff for defining ZAP-70 positivity and 98 percenthomology as the cutoff for defining a germ-line IgVH, we dividedthe 94 patients with CLL into four groups: group 1 (expressionof ZAP-70 and unmutated IgVH) included 36 patients, group 2(expression of ZAP-70 and mutated IgVH) included 10 patients,group 3 (no expression of ZAP-70 and unmutated IgVH) included1 patient, and group 4 (no expression of ZAP-70 and mutatedIgVH) included 47 patients. We found, using several algorithmsfor statistical and prediction analysis (PAM, SAM, and GeneSpring),that a signature composed of 13 mature microRNAs could discriminate(P<0.01) between group 1 and group 4, the two main groupsof patients (Table 2; and Table 1 of the Supplementary Appendix,available with the full text of this article at www.nejm.org);the prediction made using Support Vector Machine correctly classifiedall patients (Table 2 of the Supplementary Appendix). Of 13microRNAs, 9 were significantly overexpressed in group 1, thegroup with a poor prognosis (Table 2). The 10 patients in group2 were equally assigned according to their microRNA signatureto groups 1 and 4, suggesting that there are no microRNAs onthe microRNA chip that can identify distinctive characteristicsin these patients, that these two groups are not different withrespect to microRNA expression, or that this group is too smallto be correctly classified.
Table 2. MicroRNA Signature Associated with Prognostic Factors (ZAP-70 Expression and IgVH Mutations) and Disease Progression in Patients with CLL.
We applied the Support Vector Machine algorithm to an independentset of 50 samples of CLL cells with known ZAP-70 status (Table2 of the Supplementary Appendix). Using the microRNA signatureconsisting of 13 microRNAs, we found that the classificationaccording to ZAP-70 status was correct in all cases. Confirmingthe previously reported microarray specificity,20 we found thatthe signature of 13 microRNAs did not include very similar membersof the same families, such as miR-23a (one-base difference frommiR-23b) and miR-15b (four-base difference from miR-15a), whereasthe identical mature microRNAs miR-16-1 and miR-16-2 were bothpresent, indicating that the chip can discriminate between highlysimilar isoforms of microRNA.
Association between MicroRNA Expression and the Time to Initial Therapy
The treatment of CLL begins with the development of symptomaticor progressive disease, as defined according to the criteriaof the National Cancer Institute Working Group.24 Of the 94patients we studied, 41 had begun therapy (Table 1).
Using PAM survival analysis, we examined the relationship betweenthe expression of 190 microRNA genes and the time from diagnosisto initial therapy for all 94 patients. We found 9 microRNAs,all members of the 13-member prognostic signature, that bestdifferentiated patients with a short interval from diagnosisto initial therapy (average [±SD], 40±39 months)from patients with a significantly longer interval (average,88±42 months; P<0.01) (Figure 1, and Table 1 of theSupplementary Appendix). The significance of the differencesin the Kaplan–Meier curves increased if we restrictedthe analyses to the 83 patients in the two main groups (groups1 and 4) (P values decreased from <0.01 to <0.005). Allnine microRNAs that were associated with the time to initialtherapy were overexpressed, except miR-29c, in the group witha short interval from diagnosis to initial therapy (Figure 1).
Figure 1. Relationship between the Level of Expression of MicroRNA and the Time from Diagnosis to Initial Therapy.
Kaplan–Meier curves in Panel A depict the proportion of untreated patients with CLL according to whether the interval from diagnosis to therapy was short or long. In Panel B, the patients are grouped according to the level of expression of nine microRNA genes (P<0.01); the corresponding numerical data are presented in Table 3 of the Supplementary Appendix. All these genes were included in the PAM-predicted signature (available in Table 1 of the Supplementary Appendix).
Genomic Sequence Abnormalities of MicroRNA in CLL
Abnormally expressed cancer genes are frequently targets formutations that can activate or inactivate their function. Therefore,we screened 42 micro-RNAs, including 15 genes that are eithercomponents of the expression signature or members of the samegenomic clusters as the genes in the expression signature. Weidentified germ-line or somatic mutations in 11 of 75 CLL samples(15 percent) in 5 of 42 microRNAs (12 percent): miR-16-1, miR-27b,miR-29b-2, miR-187, and miR-206. None of these mutations werefound in 160 persons without cancer (P<0.001) (Table 3).All the abnormalities were in regions that are transcribed asshown by the reverse-transcriptase–polymerase-chain-reactionassay (RT-PCR) (Figure 2). Of the 11 patients with an abnormalmicroRNA sequence, 8 (73 percent) had a known personal or familyhistory of CLL or other hematopoietic or solid tumors (Table 3).
Panel A depicts the locations of mutations in microRNAs (blue) and normal nucleotide bases (red); the figure is not drawn to scale. Panel B shows the primary transcripts in B-cell CLL cells, as well as the length of the amplified genomic DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. RT+ denotes reverse transcription, RT– control without reverse transcription, and G genomic control. Panel C presents the chromatograms for the normal genome and the mutated miR-15a–miR-16-1 (CT)+7 samples. The precise position of the precursor and the location of mutation are indicated. Panel D shows the ratio of the expression levels on the basis of microRNA-microchip array (MAr) and Northern blotting (NB) for miR-16-1 and miR-15a in two pools of normal CD5+ cells and cells from both patients with the germ-line (CT)+7 mutation. The intensities of the Western blotting bands were quantified with the use of ImageQuantTL (Nonlinear Dynamics). Expression data were normalized as described in the Methods section. Data are presented as arbitrary units. Panel E shows that the germ-line mutation in pri-miR-16-1 is associated with an abnormal level of expression of the active molecule miR-16-1. Levels of expression after transfection in 293 cells of wild-type and mutant miR-16-1 and empty vector are indicated. The Northern loading normalization was performed with the use of the U6 probe, whereas the transfection levels were normalized with antibody against green fluorescent protein (GFP) on cell lysates from the same pellet as that used for Northern blotting.
In two patients, we found a CT homozygous substitution in thepri-miR-16-1, 7 bp in the 3' direction after the precursor.This genomic region is strongly conserved in all primates analyzed,25suggesting an important functional role for pri-miR-16-1. RT-PCRshowed that the pri-microRNA was at least 800 bp long and includedthe 3' region harboring the base substitution (Figure 2). MicroRNA-microchipanalysis and Northern blotting showed that CLL cells from bothpatients had a substantial reduction in the expression of miR-16-1as compared with that of normal CD5+ cells (Table 3). In mostCLL cells from these patients, we also detected a monoallelicdeletion at 13q14.3 by fluorescence in situ hybridization andloss-of-heterozygosity analysis (data not shown). This substitutionwas not found in any cells from 160 control subjects (P<0.05by the chi-square test). In both patients, normal cells fromthe buccal mucosa were heterozygous for this abnormality. Therefore,the CT change is a germ-line mutation or a very rare polymorphism;the finding that the mother and sister of one of the patientshave CLL and breast cancer, respectively, supports the existenceof a germ-line mutation. This family fulfills the minimal criteriafor familial CLL: two or more cases of CLL in first-degree relatives.26
To identify a possible molecular effect of the CT substitution,we prepared vectors containing either the wild-type allele ofthe miR-15a–miR-16-1 cluster or the mutated allele. The293 cells, which have low endogenous expression of this cluster,were transfected with the vectors. As a control we used theempty vector. The mutant transfectants expressed miR-16-1 andmiR-15a at levels that were significantly lower than those ofthe wild-type transfectants and similar to that of non-transfectedcells (Figure 2). These results indicate that the CT substitutionaffects the level of expression of mature microRNAs.
Discussion
In this study of CLL, we found a significant association betweenthe expression of certain microRNAs and the expression of ZAP-70,the mutational status of IgVH, and the time between diagnosisand initial treatment. The time from diagnosis to initial treatmentis an important factor associated with disease activity, sincetherapy for CLL is usually withheld until symptoms, advanceddisease, or both develop.27 Using a microRNA microarray composedof 190 human genes, we found a unique 13-gene molecular signatureassociated with each prognostic factor. Therefore, we believethat microRNA expression can be included in the markers withprognostic significance in CLL.
Besides its relevance as a prognostic marker, the microRNA signaturewe found may be relevant to the pathogenesis of CLL. Severalfacts provide support for its functional importance. First,all the microRNAs of this signature represent the active partsof the transcript that interact with messenger RNA, even thoughabout 20 percent of the oligomers in the microarray are specificfor pre-micro-RNA, a functionally inactive microRNA. Second,the signature consists of microRNAs that are abnormally expressedin CLL (miR-15a and miR-16-1) or other leukemias (miR-155) orare located in regions involved in cancers (miR-23b, miR-24-1,miR-29b-2, and miR-195).10,11,12,13 Third, 7 of 13 of thesemicroRNAs are members of microRNA clusters, and their levelof expression is similar, suggesting a common mechanism of generegulation that marks the differences in these two prognosticcategories of CLL samples.
The finding of mutations in two microRNA genes, miR-16-1 andmiR-15a, in CLL is important. Our previous data indicated thatmiR-16-1 and miR-15a behave as tumor suppressors in CLL. Thecombination of loss of heterozygosity plus a germ-line mutationthat we found in two patients is characteristic of the Knudsonmodel of inactivation of a tumor-suppressor gene. The presenceof pathogenic mutations in the miR-15a–miR-16-1 cluster,as well as the identification of various mutations in othermicroRNAs, indicates that this new class of genes is involvedin CLL21 and that at least some microRNAs can function as tumor-suppressorgenes.10,13,28 Because the 40 bases before and after the pre-microRNAcan influence the transcription of the microRNA,27 it is possiblethat the single-base mutation CT may affect the expression ofmicroRNA.
Most of the sequence abnormalities we identified in microRNAgenes were not found in 160 subjects without cancer, and inseveral instances, they were also found in DNA from normal cellsin the same patient. In a recent study of 96 healthy subjects,10 polymorphisms of microRNA genes were found, but none werein any of the five mutated microRNAs we found in CLL.29 SinceCLL is a disease with a frequent association in families (10to 20 percent of patients have at least one first-degree relativewith CLL) as well as other cancers,30 microRNA mutations maybe a predisposing factor for the cancers associated with CLL.Since we identified mutations in both signature-specific andsignature-independent microRNAs and we screened less than onefifth of the known microRNAs,31 the frequency of the mutationsthat we reported here (15 percent) may be an underestimate.
MicroRNAs are a recently identified class of regulatory RNAsthat function primarily by targeting specific messenger RNAs(mRNAs) for degradation or inhibition of translation and thusdecreasing the expression of the resulting protein. Our findingthat all but one of the microRNAs that predict the time to initialtherapy are overexpressed suggests that the down-regulationof target mRNAs plays a role in disease progression. Severalgenes are targeted by two different microRNAs, such as WTAP,the Wilms' tumor-1–associated protein isoform 1, whichis targeted by both miR-221 and miR-223 (Table 2). Moreover,the anti-apoptotic BCL2 gene is reported to be overexpressedin 65 to 70 percent of B-cell CLLs,32 whereas deletions or down-regulationsof miR-16-1 were reported in the same proportion of CLL samples.10We have shown that BCL2 is a target of microRNAs miR-15 andmiR-16 and that down-regulation of BCL2 protein by these microRNAstriggers apoptosis.33
In conclusion, our study shows that a unique microRNA signatureis associated with prognostic factors and disease progressionin CLL and that mutations in microRNA genes are frequent andmay have functional importance.
Supported by Program Project grants (P01CA76259, P01CA81534,and P30CA56036, to Drs. Kipps and Croce ) from the NationalCancer Institute, by a Kimmel Scholar award (to Dr. Calin),and by grants from the Italian Ministry of Public Health, theItalian Ministry of University Research, and the Italian Associationfor Cancer Research (to Drs. Negrini and Volinia).
Source Information
From the Department of Molecular Virology, Immunology, and Medical Genetics and Comprehensive Cancer Center, Ohio State University, Columbus (G.A.C., A.C., G.D.L., M.S., S.E.W., M.V.I., R.V., N.I.S., M.F., R.I., T.P., F.P., C.R., H.A., S.V., C.L., C.M.C.); the Department of Experimental and Diagnostic Medicine, Interdepartment Center for Cancer Research, University of Ferrara, Ferrara, Italy (M.F., M.N.); the Kimmel Cancer Center, Thomas Jefferson University, Philadelphia (R.G., C.S.); and the Department of Medicine, University of California, San Diego, La Jolla (L.R., T.J.K.).
Address reprint requests to Dr. Croce at Ohio State University, Comprehensive Cancer Center, Wiseman Hall, Rm. 385K, 400 12th Ave., Columbus, OH 43210, or at carlo.croce{at}osumc.edu.
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(2008). Genomic aberrations in mantle cell lymphoma detected by interphase fluorescence in situ hybridization. Incidence and clinicopathological correlations. haematol
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(2007). Microarray analysis of microRNA expression in peripheral blood cells of systemic lupus erythematosus patients. Lupus
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(2007). Assessment of Chronic Lymphocytic Leukemia and Small Lymphocytic Lymphoma by Absolute Lymphocyte Counts in 2,126 Patients: 20 Years of Experience at The University of Texas M.D. Anderson Cancer Center. JCO
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(2007). Tumor protein 53-induced nuclear protein 1 expression is repressed by miR-155, and its restoration inhibits pancreatic tumor development. Proc. Natl. Acad. Sci. USA
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A correction has been published: N Engl J Med 2006;355(5):533.
T)+7 substitution, by ligating the relevant open reading frame in a sense orientation into a mammalian expression vector pSR-GFP-Neo (OligoEngine). All sequenced constructs were transfected in 293 cells with the use of Lipofectamine2000 according to the manufacturer's protocol (Invitrogen). The expression of both constructs was assessed by Northern blotting. 
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