Proteomic Analysis of Chronic Lymphocytic Leukemia Subtypes with Mutated or Unmutated Ig VH Genes

Abstract

Chronic lymphocytic leukemia (CLL) is a common hematopoietic malignant disease with variable outcome. CLL has been divided into distinct groups based on whether somatic hypermutation has occurred in the variable region of the immunoglobulin heavy-chain locus or alternatively if the cells express higher levels of the CD38 protein. We have analyzed the proteome of 12 cases of CLL (six mutated (M-CLL) and six unmutated (UM-CLL) immunoglobulin heavy-chain loci; seven CD38-negative and five CD38-positive) using two-dimensional electrophoresis and mass spectrometry. Statistical evaluation using principal component analysis indicated significant differences in patterns of protein expression between the cases with and without somatic mutation. Specific proteins indicated by principal component analysis as varying between the prognostic groups were characterized using mass spectrometry. The levels of F-actin-capping protein β subunit, 14-3-3 β protein, and laminin-binding protein precursor were significantly increased in M-CLL relative to UM-CLL. In addition, primary sequence data from tandem mass spectrometry showed that nucleophosmin was present as several protein spots in M-CLL but was not detected in UM-CLL samples, suggesting that several post-translationally modified forms of nucleophosmin vary between these two sample groups. No specific differences were found between CD38-positive and -negative patient samples using the same approach. The results presented show that proteomic analysis can complement other approaches in identifying proteins that may have potential value in the biological and diagnostic distinction between important clinical subtypes of CLL. Chronic lymphocytic leukemia (CLL) is a common hematopoietic malignant disease with variable outcome. CLL has been divided into distinct groups based on whether somatic hypermutation has occurred in the variable region of the immunoglobulin heavy-chain locus or alternatively if the cells express higher levels of the CD38 protein. We have analyzed the proteome of 12 cases of CLL (six mutated (M-CLL) and six unmutated (UM-CLL) immunoglobulin heavy-chain loci; seven CD38-negative and five CD38-positive) using two-dimensional electrophoresis and mass spectrometry. Statistical evaluation using principal component analysis indicated significant differences in patterns of protein expression between the cases with and without somatic mutation. Specific proteins indicated by principal component analysis as varying between the prognostic groups were characterized using mass spectrometry. The levels of F-actin-capping protein β subunit, 14-3-3 β protein, and laminin-binding protein precursor were significantly increased in M-CLL relative to UM-CLL. In addition, primary sequence data from tandem mass spectrometry showed that nucleophosmin was present as several protein spots in M-CLL but was not detected in UM-CLL samples, suggesting that several post-translationally modified forms of nucleophosmin vary between these two sample groups. No specific differences were found between CD38-positive and -negative patient samples using the same approach. The results presented show that proteomic analysis can complement other approaches in identifying proteins that may have potential value in the biological and diagnostic distinction between important clinical subtypes of CLL. Chronic lymphocytic leukemia (CLL) 1The abbreviations used are: CLL, chronic lymphocytic leukemia; Ig VH, immunoglobulin heavy-chain locus; M-CLL, CLL with somatic mutation of Ig VH; UM-CLL, CLL without somatic mutation of Ig VH; MALDI-ToF, matrix-assisted laser desorption ionization time-of-flight; PCA, principal component analysis; 2D, two-dimensional; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. is the most common adult B-cell malignancy in the Western world (1Caligaris-Cappio F. Hamblin T.J. B-cell chronic lymphocytic leukemia: a bird of a different feather.J. Clin. Oncol. 1999; 17: 399-408Google Scholar). CLL cells share particular immunophenotypic features (expression of mature B-cell markers, CD23, CD5, and weak expression of surface immunoglobulin) and demonstrate a pattern of expression of a range of genes that suggests the neoplastic cells are related to memory B-lymphocytes (2Konikova E. Babusikova O. Mesarosova A. Kusenda J. Glasova M. Cytoplasmic and surface membrane phenotypic markers in cells of B cell chronic lymphocytic leukemia.Neoplasma (Bratisl.). 1994; 41: 69-74Google Scholar, 3Pangalis G.A. Angelopoulou M.K. Vassilakopoulos T.P. Siakantaris M.P. Kittas C. B-chronic lymphocytic leukemia, small lymphocytic lymphoma, and lymphoplasmacytic lymphoma, including Waldenstrom's macroglobulinemia: a clinical, morphologic, and biologic spectrum of similar disorders.Semin. Hematol. 1999; 36: 104-114Google Scholar). However, in different cases of the disease, there may be considerable variation of clinical behavior and of prognosis. The clinical course of CLL is heterogeneous; some patients progress rapidly to early death, whereas others exhibit a more stable disease lasting many years. A considerable research effort has therefore been directed toward the identification of markers to guide effective consideration of treatment. In this regard, recently it has been shown that CLL may be divided into clinically distinct groups depending on whether the variable region of the immunoglobulin heavy-chain locus (Ig VH) has undergone somatic mutation. Cases of CLL with somatic mutation of Ig VH have a more favorable prognosis than those where Ig VH has not undergone somatic mutation (4Damle R.N. Wasil T. Fais F. Ghiotto F. Valetto A. Allen S.L. Buchbinder A. Budman D. Dittmar K. Kolitz J. Lichtman S.M. Schulman P. Vinciguerra V.P. Rai K.R. Ferrarini M. Chiorazzi N. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia.Blood. 1999; 94: 1840-1847Google Scholar, 5Hamblin T.J. Davis Z. Gardiner A. Oscier D.G. Stevenson F.K. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia.Blood. 1999; 94: 1848-1854Google Scholar, 6Maloum K. Davi F. Merle-Beral H. Pritsch O. Magnac C. Vuillier F. Dighiero G. Troussard X. Mauro F.F. Benichou J. Expression of unmutated VH genes is a detrimental prognostic factor in chronic lymphocytic leukemia.Blood. 2000; 96: 377-379Google Scholar). Another potential prognostic marker in CLL is expression of CD38 on the cell surface where increased expression was indicated to be a marker for poor prognosis (4Damle R.N. Wasil T. Fais F. Ghiotto F. Valetto A. Allen S.L. Buchbinder A. Budman D. Dittmar K. Kolitz J. Lichtman S.M. Schulman P. Vinciguerra V.P. Rai K.R. Ferrarini M. Chiorazzi N. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia.Blood. 1999; 94: 1840-1847Google Scholar, 7Hamblin T.J. Orchard J.A. Gardiner A. Oscier D.G. Davis Z. Stevenson F.K. Immunoglobulin V genes and CD38 expression in CLL.Blood. 2000; 95: 2455-2457Google Scholar, 8Durig J. Naschar M. Schmucker U. Renzing-Kohler K. Holter T. Huttmann A. Duhrsen U. CD38 expression is an important prognostic marker in chronic lymphocytic leukaemia.Leukemia (Baltimore). 2002; 16: 30-35Google Scholar, 9D'Arena G. Musto P. Cascavilla N. Dell'Olio M. Di Renzo N. Perla G. Savino L. Carotenuto M. CD38 expression correlates with adverse biological features and predicts poor clinical outcome in B-cell chronic lymphocytic leukemia.Leuk. Lymphoma. 2001; 42: 109-114Google Scholar, 10Chevallier P. Penther D. Avet-Loiseau H. Robillard N. Ifrah N. Mahe B. Hamidou M. Maisonneuve H. Moreau P. Jardel H. Harousseau J.L. Bataille R. Garand R. CD38 expression and secondary 17p deletion are important prognostic factors in chronic lymphocytic leukaemia.Br. J. Haematol. 2002; 116: 142-150Google Scholar). CD38 level has been suggested to correlate with unmutated Ig VH gene status, but more recent data suggests CD38 expression to be independently variable from VH gene status (11Hamblin T.J. Orchard J.A. Ibbotson R.E. Davis Z. Thomas P.W. Stevenson F.K. Oscier D.G. CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease.Blood. 2002; 99: 1023-1029Google Scholar, 12Vilpo J. Tobin G. Hulkkonen J. Hurme M. Thunberg U. Sundstrom C. Vilpo L. Rosenquist R. Surface antigen expression and correlation with variable heavy-chain gene mutation status in chronic lymphocytic leukemia.Eur. J. Haematol. 2003; 70: 53-59Google Scholar, 13Jelinek D.F. Tschumper R.C. Geyer S.M. Bone N.D. Dewald G.W. Hanson C.A. Stenson M.J. Witzig T.E. Tefferi A. Kay N.E. Analysis of clonal B-cell CD38 and immunoglobulin variable region sequence status in relation to clinical outcome for B-chronic lymphocytic leukaemia.Br. J. Haematol. 2001; 115: 854-861Google Scholar, 14Thunberg U. Johnson A. Roos G. Thorn I. Tobin G. Sallstrom J. Sundstrom C. Rosenquist R. CD38 expression is a poor predictor for VH gene mutational status and prognosis in chronic lymphocytic leukemia.Blood. 2001; 97: 1892-1894Google Scholar). Thus, VH gene status and CD38 expression appear to be independent prognostic markers for CLL. Nonetheless, there is still a requirement for effective prognostic indicators in CLL that can rapidly enable treatment strategies to be mapped out. Microarray analysis of CLL patients is an obvious and potentially beneficial means of defining specific prognostic indicators in CLL. Genes whose expression levels significantly correlate with patient survival and/or with clinical staging have been found and categorized as encoding or potentiating activity of cell adhesion molecules (15Stratowa C. Loffler G. Lichter P. Stilgenbauer S. Haberl P. Schweifer N. Dohner H. Wilgenbus K.K. CDNA microarray gene expression analysis of B-cell chronic lymphocytic leukemia proposes potential new prognostic markers involved in lymphocyte trafficking.Int. J. Cancer. 2001; 91: 474-480Google Scholar). It remains to be seen if these proteins or their gene transcripts have value as disease markers. The transcriptomes of UM-CLL and M-CLL have been compared, and analysis has revealed differential expression of a relatively small number of mRNA species between the two CLL subtypes (23 transcribed genes of 12,000 genes analyzed). The restricted number of genes was able to distinguish mutated versus unmutated cases. However, a transcriptomics approach may not alone be sufficient to distinguish important differences between UM-CLL and M-CLL. For example, although CD38 expression has been shown to correlate with poor prognosis CLL, there is no change in CD38 at the level of the transcriptome (16Rosenwald A. Alizadeh A.A. Widhopf G. Simon R. Davis R.E. Yu X. Yang L. Pickeral O.K. Rassenti L.Z. Powell J. Botstein D. Byrd J.C. Grever M.R. Cheson B.D. Chiorazzi N. Wilson W.H. Kipps T.J. Brown P.O. Staudt L.M. Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia.J. Exp. Med. 2001; 194: 1639-1647Google Scholar). This may be because the level of transcription of a specific gene does not directly correlate with the level of expression of its protein product within the cell. Differences between gene transcription and protein expression are the result of post-transcriptional regulation, including altered mRNA translation, and protein stability (17Gygi S.P. Rochon Y. Franza B.R. Aebersold R. Correlation between protein and mRNA abundance in yeast.Mol. Cell. Biol. 1999; 19: 1720-1730Google Scholar). In neoplastic cells additional mechanisms include proteasome-mediated degradation of specific proteins induced by leukemogenic oncogenes (18Dai Z. Quackenbush R.C. Courtney K.D. Grove M. Cortez D. Reuther G.W. Pendergast A.M. Oncogenic Abl and Src tyrosine kinases elicit the ubiquitin-dependent degradation of target proteins through a Ras-independent pathway.Genes Dev. 1998; 12: 1415-1424Google Scholar). For these reasons, analysis of protein expression is both complementary and additive to the data obtained by transcriptomic analysis. Here we have asked whether proteomic analysis reveals specific and potentially valuable diagnostic indicators for UM-CLL compared with M-CLL and/or for CD38-positive compared with CD38-negative CLL patient samples. Using principal component analysis (PCA) of 2D gels we have shown differential expression of proteins between UM-CLL and M-CLL samples, identifying potential marker proteins associated with these different CLL subtypes and thereby revealing potential value in this approach to the characterization of leukemia subtypes. Following Southampton and Southwest Hampshire local research ethics committee approval, peripheral blood was obtained from 12 patients with classical B-cell CLL. Peripheral blood mononuclear cells were isolated by Ficoll-Paque gradient centrifugation (Amersham Biosciences), washed, and cryopreserved. CLL cells were not further purified to minimize in vitro manipulations. Patient samples were characterized for Ig VH gene mutation status and CD38 expression (5Hamblin T.J. Davis Z. Gardiner A. Oscier D.G. Stevenson F.K. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia.Blood. 1999; 94: 1848-1854Google Scholar). Patients were selected to provide a representative selection of VH gene and CD38 status (Table I). Of these 12 patients, seven patients were CD38-negative, and five were CD38-positive; there were six each of UM-CLL and M-CLL in respect to Ig VH gene status. All cases scored 4 or 5 using the for CLL and were of A or B using Dighiero G. T. J.L. of to the of Scholar). Patient are shown in I. analysis was on six UM-CLL and six M-CLL samples and of CLL VH gene VH VH VH VH VH VH VH VH 12 VH VH VH VH in a new were from patient mononuclear cell was in of CHAPS, (Amersham The samples were at 12,000 for at was using gradient and the (Amersham Biosciences), for a of at The was a using the (Amersham were for in and for in the same gels were gels of were with using the of A. M. O. M. of proteins Scholar). For mass gels of from 5 cells were and the proteins were with were in in of a and in for to of at were to for 4 The gels were and the were as for analysis using was used for and for the a for the based on patient This was selected on the of the most spots and a for the analysis. gels were for each of the UM-CLL, M-CLL, and CD38-negative The gels are a of several gels to a that has and associated variation within the groups were for pattern The for in the were that be present on five of six Following the the was to features as and the that been The and gels were to the and were to for in by the were to their and to the using the and been the gels were analyzed for protein of were from the by and with modified as A. M. O. M. of proteins Scholar, U. C. J. of an for the of protein for Scholar, F. S.M. identification of proteins from a for the of to 1999; Scholar). for matrix-assisted laser desorption ionization analysis were by a small of the with an of a of in mass was on a mass All mass were with or was for protein using mass for each was based on at five an and value as from the CLL 2D and a value of within the that the mass was from the protein in the The clinical of the samples obtained from patients with CLL are shown in I. 12 samples were because six samples with unmutated Ig VH and six samples with mutated Ig within this of samples, five samples that the prognostic marker CD38 were for with seven samples with levels of CD38 CD38 and Ig VH are to be independent prognostic markers. was to samples as within the of samples UM-CLL versus M-CLL and CD38-positive versus The was to potential prognostic protein markers with of these markers. samples from six cases of M-CLL and six of UM-CLL were analyzed using two-dimensional were between 4 and to a level of and were two were for samples, and similar protein patterns were protein spots were detected on the gels from the patient to the of gels from different patients, a of differences between was not based on were therefore for analysis within the was to spots and on a as A representative is shown in and is to show the of spots and by mass for of for and was using the The proteins are in of proteins from B-cell CLL patient protein V small protein protein β factor protein antigen protein protein factor β protein protein protein protein B protein protein protein β protein to in a new For the was to a value of the In to the from CLL samples, level of gels can be from groups of gels to the of distinct within the Thus, gels were for the M-CLL and UM-CLL gels and for the CD38-positive and CD38-negative these was between the and the protein expression were compared for each with microarray that show at the mRNA we found although not in protein expression of the gels of the was to further the level of between the UM-CLL and M-CLL gels and rapidly to protein spots that to potential differences between the two groups. is a analysis that a number of potentially variables into a number of variables principal is a This of analysis the of a of data and has been to be of value in proteomic analysis H. M. G. P. S. A. N. M. approach in the of the activity of the new 2000; Scholar). It can be used to of protein spots and potentially to proteins that may have prognostic The results of this analysis are presented in the of the protein spots in the patient groups using a 2D based on Thus, the within the 2D gels analyzed using can between the UM-CLL and M-CLL samples by those spots that the We additional analysis of for the potential the relative abundance of those protein spots shown by to between UM-CLL and M-CLL. that showed using are shown in and of their expression levels are presented in The of those is shown in The others show some of but the were using the or the as significantly altered between M-CLL and UM-CLL are the F-actin-capping protein β subunit, 14-3-3 β protein, and laminin-binding precursor protein. these proteins the and in the region where patients The and of protein spots from spots were used to protein of CD38-positive and versus CD38-negative and patient gels not protein spots the protein spots that between M-CLL and UM-CLL not show significant change in respect to the CD38 for M-CLL the of The of protein spots that in expression between M-CLL and UM-CLL are were and by mass of their and are shown in In to those proteins whose level of expression between M-CLL and UM-CLL, the two clinical groups were by the or of of The protein was present in the M-CLL samples but not in the UM-CLL samples spots This region was to be a between the two sample using and analysis and It was shown to be a using the analysis of not analysis of the spots within this region revealed a similar mass spectrum for suggested the spots forms of the same protein. the spots as forms of the protein were not in UM-CLL samples. The of this was to potentially and valuable prognostic indicators that correlate with CD38 expression and/or Ig VH mutation status. of identification of in many protein levels between 2D analysis the identification of differences between patient sample in several of gels the patient samples of a particular relatively progress to be in data analysis. analysis of these data using as identification of spots that vary between sample the of other features as for the samples means that it is relatively to and the of an between sample This of was using CLL patient based on Ig VH mutation status or CD38 expression using the gels for the UM-CLL and M-CLL patients a of proteins for further proteins were for relative expression level in each sample and This analysis of relative between M-CLL and UM-CLL revealed the of each sample to the differences within the that this approach not the from spots were and we found no other The between the 2D patterns of UM-CLL and M-CLL seen in these the results from microarray analysis of the CLL samples that that the two groups have similar activity and memory U. Y. G.A. M. G. H. A. G. L. L. A. A. R. expression of B cell chronic lymphocytic leukemia reveals a phenotype related to memory B Exp. Med. 2001; 194: Scholar). prognostic indicators are still to and further the clinically variable course of CLL. by the proteomic approaches in this are those with relatively we therefore that the proteins may have value in the diagnostic distinction between the somatic mutated and unmutated subtypes of CLL. proteins were that to differences between M-CLL and UM-CLL. F-actin-capping protein β is at higher levels in M-CLL compared with UM-CLL. This protein a in the of the It is that other molecules to were found to have value between UM-CLL and M-CLL in the indicated a significant between the groups in relation to the expression of the laminin-binding protein The laminin-binding protein precursor is a precursor of the but has a range of in its precursor these include the of D. S. precursor factor and Biol. 2003; and the and to the of molecules that cell and cell survival H. K. T. T. T. to protein to of the 2001; Scholar). It is to that two distinct forms of as potential value and in analysis showed that the of these forms between the two clinical groups of CLL, a that is with altered of molecules within the clinical this to F. L. L. G. F. is in the cells of B chronic lymphocytic leukemia and cell leukemia.Blood. Scholar). of altered expression of the proteins in the mutated and unmutated of CLL may altered activity between those of are with of CLL adhesion and as that by A.M. J.C. J. in chronic lymphocytic leukemia.Blood. that a similar expression of adhesion cases of CLL with an in both and may have significant clinical because in other microarray the transcription of factors adhesion was to the clinical of CLL, suggesting that increased adhesion may be associated with more aggressive CLL (15Stratowa C. Loffler G. Lichter P. Stilgenbauer S. Haberl P. Schweifer N. Dohner H. Wilgenbus K.K. CDNA microarray gene expression analysis of B-cell chronic lymphocytic leukemia proposes potential new prognostic markers involved in lymphocyte trafficking.Int. J. Cancer. 2001; 91: 474-480Google Scholar, O. Y. M. M. molecules expression in cells from and and chronic lymphocytic leukemia (CLL) 2001; Scholar). microarray have shown unmutated cases of CLL to have an phenotype D.F. Tschumper R.C. Geyer S.M. Bone N.D. Dewald G.W. Hanson C.A. Stenson M.J. Witzig T.E. Tefferi A. Kay N.E. Analysis of clonal B-cell CD38 and immunoglobulin variable region sequence status in relation to clinical outcome for B-chronic lymphocytic leukaemia.Br. J. Haematol. 2001; 115: 854-861Google Scholar). In respect to the protein nucleophosmin the of this protein many of as to it may to differences between the subtypes of CLL. is a that between the and potentially the between nucleophosmin and directly with to its stability and and is for E. J.C. D. B. the stability and activity of Biol. 2002; Scholar). or mutation of in CLL is associated with disease and poor prognosis I. S. Mauro S. O. T. C. F. S. A. F. R. expression in B-cell chronic lymphocytic leukemia: a marker of disease and poor 1998; 91: Scholar, D.G. Gardiner S. Davis Ibbotson R.E. Thomas P.W. J.A. Orchard J.A. Hamblin T.J. analysis of prognostic factors in clinical gene mutational status, and or mutation of the gene are independent prognostic 2002; Scholar). Thus, the of a of nucleophosmin spots in M-CLL cells may to stability and in those cases and may to their clinical outcome. The increased level of 14-3-3 β protein in M-CLL compared with UM-CLL may be in this 14-3-3 proteins are and proteins involved in and proteins in a to the Src and distinct from and proteins because 14-3-3 proteins as of their H. 14-3-3 and 2000; Scholar, G. J. 14-3-3 new to 2001; Scholar). In this proteomic analysis has for further in respect to the differences between and M-CLL samples that may to differential prognosis or may be as markers for disease outcome. We have a to differences between cell sample groups that may be more In this we have that proteomic analysis has a potentially valuable in the of clinical results obtained by are both complementary and additive to the results of microarray analysis (16Rosenwald A. Alizadeh A.A. Widhopf G. Simon R. Davis R.E. Yu X. Yang L. Pickeral O.K. Rassenti L.Z. Powell J. Botstein D. Byrd J.C. Grever M.R. Cheson B.D. Chiorazzi N. Wilson W.H. Kipps T.J. Brown P.O. Staudt L.M. Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia.J. Exp. Med. 2001; 194: 1639-1647Google Scholar).