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Analysis of Signal Transducer and Activator of Transcription 3 (Stat 3) Pathway in Multiple Myeloma

2003; Elsevier BV; Volume: 162; Issue: 5 Linguagem: Inglês

10.1016/s0002-9440(10)64278-2

1525-2191

Leticia Quintanilla‐Martínez, Marcus Kremer, Katja Specht, Julia Calzada‐Wack, Michaela Nathrath, Robert Schaich, Heinz Höfler, Falko Fend,

NF-κB Signaling Pathways

The signal transducer and activator of transcription molecules (Stats) play key roles in cytokine-induced signal transduction. Recently, it was proposed that constitutively activated Stat 3 (Stat 3 phosphorylated) contributes to the pathogenesis of multiple myeloma (MM) by preventing apoptosis and inducing proliferation. The study aim was to investigate Stat 3 activation in a series of multiple myeloma (MM) cases and its effect on downstream targets such as the anti-apoptotic proteins Bcl-xL, Mcl-1, and Bcl-2, and the cell-cycle protein cyclin D1. Forty-eight cases of MM were analyzed. Immunohistochemistry was performed on paraffin sections using antibodies against cyclin D1, Bcl-2, Bcl-xL, Mcl-1, p21, Stat 3, and Stat 3 phosphorylated (P). Their specificity was corroborated by Western blot analysis using eight human MM cell lines as control. The proliferation rate was assessed with the antibody MiB1. In addition, the mRNA levels of cyclin D1 and Stat 3 were determined by quantitative real-time reverse transcriptase-polymerase chain reaction of paraffin-embedded microdissected tissue. Three different groups determined by the expression of Stat 3P and cyclin D1 (protein and mRNA) were identified: group 1, Stat 3-activated (23 cases, 48%). All cases revealed nuclear expression of Stat 3P. No elevation of Stat 3 mRNA was identified in any of the cases. Three cases in this group showed intermediate to low cyclin D1 protein and mRNA expression. Group 2 included 15 (31%) cases with cyclin D1 staining and lack of Stat 3P. All cases showed intermediate to high levels of cyclin D1 mRNA expression. Group 3 included 10 (21%) cases with no expression of either cyclin D1 or Stat 3P. High levels of anti-apoptotic proteins Bcl-xL and Mcl-1 were identified in 89% and 100% of all cases, respectively. In contrast to Bcl-xL and Mcl-1, the expression of Bcl-2 showed an inverse correlation with proliferation rate (P: 0.0003). No significant differences were found between the three groups in terms of proliferation rate or expression of anti-apoptotic proteins. However, cyclin D1+ cases were always well differentiated and were more likely to show a lymphoplasmocytoid differentiation (chi-square = 9.55). Overall, constitutive activation of Stat 3 was found in almost half (48%) of the investigated MM cases. However, this does not seem to have a major impact on the expression of anti-apoptotic proteins and proliferation. We showed that cyclin D1 overexpression and Stat 3 activation are, mutually exclusive events in MM (P = 0.0066). The universal expression of Mcl-1, independent of activated Stat 3, suggests that its expression is constitutive and that it might play an important role in the pathogenesis of MM. The signal transducer and activator of transcription molecules (Stats) play key roles in cytokine-induced signal transduction. Recently, it was proposed that constitutively activated Stat 3 (Stat 3 phosphorylated) contributes to the pathogenesis of multiple myeloma (MM) by preventing apoptosis and inducing proliferation. The study aim was to investigate Stat 3 activation in a series of multiple myeloma (MM) cases and its effect on downstream targets such as the anti-apoptotic proteins Bcl-xL, Mcl-1, and Bcl-2, and the cell-cycle protein cyclin D1. Forty-eight cases of MM were analyzed. Immunohistochemistry was performed on paraffin sections using antibodies against cyclin D1, Bcl-2, Bcl-xL, Mcl-1, p21, Stat 3, and Stat 3 phosphorylated (P). Their specificity was corroborated by Western blot analysis using eight human MM cell lines as control. The proliferation rate was assessed with the antibody MiB1. In addition, the mRNA levels of cyclin D1 and Stat 3 were determined by quantitative real-time reverse transcriptase-polymerase chain reaction of paraffin-embedded microdissected tissue. Three different groups determined by the expression of Stat 3P and cyclin D1 (protein and mRNA) were identified: group 1, Stat 3-activated (23 cases, 48%). All cases revealed nuclear expression of Stat 3P. No elevation of Stat 3 mRNA was identified in any of the cases. Three cases in this group showed intermediate to low cyclin D1 protein and mRNA expression. Group 2 included 15 (31%) cases with cyclin D1 staining and lack of Stat 3P. All cases showed intermediate to high levels of cyclin D1 mRNA expression. Group 3 included 10 (21%) cases with no expression of either cyclin D1 or Stat 3P. High levels of anti-apoptotic proteins Bcl-xL and Mcl-1 were identified in 89% and 100% of all cases, respectively. In contrast to Bcl-xL and Mcl-1, the expression of Bcl-2 showed an inverse correlation with proliferation rate (P: 0.0003). No significant differences were found between the three groups in terms of proliferation rate or expression of anti-apoptotic proteins. However, cyclin D1+ cases were always well differentiated and were more likely to show a lymphoplasmocytoid differentiation (chi-square = 9.55). Overall, constitutive activation of Stat 3 was found in almost half (48%) of the investigated MM cases. However, this does not seem to have a major impact on the expression of anti-apoptotic proteins and proliferation. We showed that cyclin D1 overexpression and Stat 3 activation are, mutually exclusive events in MM (P = 0.0066). The universal expression of Mcl-1, independent of activated Stat 3, suggests that its expression is constitutive and that it might play an important role in the pathogenesis of MM. Signal transducer and activator of transcription (Stat) 3 is a cytoplasmic latent transcription factor that becomes activated by phosphorylation, typically in response to extracellular ligands such as interleukin (IL)-6, platelet-derived growth factor, or epidermal growth factor.1Darnell Jr, JE STATs and gene regulation.Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3401) Google Scholar Specifically, IL-6 binds to its α chain receptor and induces homodimerization of gp 130 and activation of the intracytoplasmic Janus family of tyrosine kinases (Jaks), with downstream signaling via the Stat- or Ras-dependent mitogen-activated protein kinase cascades. Once phosphorylated by Janus kinases, Stat 3 dimerizes and translocates to the nucleus, where it activates the transcriptionof target genes. Stat 3 activation has been implicated in the regulation of cell proliferation, differentiation, and apoptosis.2Bromberg J Darnell Jr, JE The role of STATs in transcriptional control and their impact on cellular function.Oncogene. 2000; 19: 2468-2473Crossref PubMed Scopus (1051) Google Scholar The importance of Stat 3 gene is highlighted by the fact that its disruption in animal models causes embryonic lethality.3Takeda K Noguchi K Shi W Tanaka T Matsumoto M Yoshida N Kishimoto T Akira S Targeted disruption of the mouse Stat3 gene leads to embryonic lethality.Proc Natl Acad Sci USA. 1997; 94: 3801-3804Crossref PubMed Scopus (1109) Google Scholar In the last years, several studies have shown that tumor cell lines and samples derived from human cancers, including breast, hematopoietic, head and neck, lung, kidney, prostate, and ovarian cancers frequently express activated or phosphorylated Stat 3 (Stat 3P),4O'Shea JJ Gadina M Schreiber RD Cytokine signaling in 2002: new surprises in the Jak/Stat pathway.Cell. 2002; 109: 121-131Abstract Full Text Full Text PDF Scopus (953) Google Scholar suggesting that Stat 3 plays a critical role in regulating fundamental processes associated with malignant transformation and cell survival.5Bowman T Garcia R Turkson J Jove R STATs in oncogenesis.Oncogene. 2000; 19: 2474-2488Crossref PubMed Scopus (1593) Google Scholar Accordingly, recent in vitro data demonstrated that a constitutively active form of Stat 3 was sufficient to produce anchorage-independent cell proliferation and tumor formation in nude mice, thus emphasizing its oncogenic potential.6Bromberg JF Wrzeszczynska MH Devgan G Zhao Y Pestell RG Albanese C Darnell Jr, JE Stat3 as an oncogene.Cell. 1999; 98: 295-303Abstract Full Text Full Text PDF PubMed Scopus (2517) Google Scholar However, the biological mechanisms by which Stat 3 contributes to oncogenesis are not completely understood. Critical Stat 3-regulated genes proposed to be involved in the oncogenic process are cyclin D1, c-myc, and Bcl-xL, whose mRNA levels were found threefold to fivefold up-regulated as a consequence of Stat 3 activation.6Bromberg JF Wrzeszczynska MH Devgan G Zhao Y Pestell RG Albanese C Darnell Jr, JE Stat3 as an oncogene.Cell. 1999; 98: 295-303Abstract Full Text Full Text PDF PubMed Scopus (2517) Google Scholar Recently, Catlett-Falcone and colleagues,7Catlett-Falcone R Landowski TH Oshiro MM Turkson J Levitzki A Savino R Ciliberto G Moscinski L Fernandez-Luna JL Nuñez G Dalton WS Jove R Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells.Immunity. 1999; 10: 105-115Abstract Full Text Full Text PDF PubMed Scopus (1455) Google Scholar reported that constitutively activated Stat 3 is expressed in U266 myeloma cells and in the neoplastic cells of most patients with multiple myeloma (MM), in a third of them at very high levels. They presented evidence that constitutive Stat 3P induces Bcl-xL expression and confers resistance to Fas-induced apoptosis in U266 cells. Another anti-apoptotic member of the Bcl-2 family induced by IL-6 in a Stat 3-dependent way, myeloid cell factor 1 (Mcl-1), also has been reported to be a critical survival factor for MM.8Puthier D Bataille R Amiot M IL-6 up-regulates Mcl-1 in human myeloma cells through JAK/STAT rather than ras/MAP kinase pathway.Eur J Immunol. 1999; 29: 3945-3950Crossref PubMed Scopus (226) Google Scholar, 9Zhang B Gojo I Fenton RG Myeloid cell factor-1 is a critical survival factor for multiple myeloma.Blood. 2002; 99: 1885-1893Crossref PubMed Scopus (351) Google Scholar, 10Puthier D Derenne S Barille S Moreau P Harousseau JL Bataille R Amiot M Mcl-1 and Bcl-xl are co-regulated by IL-6 in human myeloma cells.Br J Haematol. 1999; 107: 392-395Crossref PubMed Scopus (136) Google Scholar The deregulated expression of Bcl-xL and Mcl-1 in MM is of particular interest because MM is a B-cell neoplasia characterized by accumulation of slowly proliferating malignant plasma cells, and the up-regulation of anti-apoptotic genes is thought to play an important role in the pathogenesis of this disorder.11Hallek M Bergsagel PL Anderson KC Multiple myeloma: increasing evidence for a multistep transformation process.Blood. 1998; 91: 3-21Crossref PubMed Google Scholar However, activated Stat 3 induces also genes that are involved in the control of cell cycle progression and proliferation such as cyclin D1 and c-myc. Cyclin D1 protein is overexpressed in 25 to 30% of the MM cases, presumably as a consequence of the t(11;14).12Pruneri G Fabris S Balsini L Carboni N Zagano S Colombi MA Ciceri G Lombardi L Rocchi M Buffa R Maiolo AT Neri A Immunohistochemical analysis of cyclin D1 shows deregulated expression in multiple myeloma with the t(11;14).Am J Pathol. 2000; 156: 1505-1513Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 13Wilson CS Butch AW Lai R Medeiros J Sawyer JR Barlogie B McCourty A Kelly K Brynes RK Cyclin D1 and E2F-1 immunoreactivity in bone marrow biopsy specimens of multiple myeloma: relationship to proliferative activity, cytogenetic abnormalities and DNA ploidy.Br J Haematol. 2001; 112: 776-782Crossref PubMed Scopus (28) Google Scholar, 14Hoyer JD Hanson CA Fonseca R Greipp PR Dewald GW Kurtin PJ The (11;14)(q13;q32) translocation in multiple myeloma. A morphologic and immunohistochemical study.Am J Clin Pathol. 2000; 113: 831-837Crossref PubMed Scopus (109) Google Scholar, 15Vasef MA Medeiros LJ Yospur LS Sun NC McCourty A Brynes RK Cyclin D1 protein in multiple myeloma and plasmacytoma: an immunohistochemical study using fixed, paraffin-embedded tissue sections.Mod Pathol. 1997; 10: 927-932PubMed Google Scholar However, the t(11;14) is identified in only 4 to 10% of bone marrow specimens when examined by conventional cytogenetics, and in 15 to 20% using fluorescent in situ hybridization.12Pruneri G Fabris S Balsini L Carboni N Zagano S Colombi MA Ciceri G Lombardi L Rocchi M Buffa R Maiolo AT Neri A Immunohistochemical analysis of cyclin D1 shows deregulated expression in multiple myeloma with the t(11;14).Am J Pathol. 2000; 156: 1505-1513Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 16Avet-Loiseau H Facon T Grosbois B Magrangeas F Rapp M-J Harousseau J-L Minvielle S Bataille R for the intergroupe Francophone du Myelome Oncogenesis of multiple myeloma: 14q32 and 13q chromosomal abnormalities are not randomly distributed, but correlate with natural history, immunological features, and clinical presentation.Blood. 2002; 99: 2185-2191Crossref PubMed Scopus (311) Google Scholar Therefore, in a proportion of MM cases with dysregulated cyclin D1 no apparent molecular abnormalities of the bcl-1 locus are identified.12Pruneri G Fabris S Balsini L Carboni N Zagano S Colombi MA Ciceri G Lombardi L Rocchi M Buffa R Maiolo AT Neri A Immunohistochemical analysis of cyclin D1 shows deregulated expression in multiple myeloma with the t(11;14).Am J Pathol. 2000; 156: 1505-1513Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 13Wilson CS Butch AW Lai R Medeiros J Sawyer JR Barlogie B McCourty A Kelly K Brynes RK Cyclin D1 and E2F-1 immunoreactivity in bone marrow biopsy specimens of multiple myeloma: relationship to proliferative activity, cytogenetic abnormalities and DNA ploidy.Br J Haematol. 2001; 112: 776-782Crossref PubMed Scopus (28) Google Scholar, 14Hoyer JD Hanson CA Fonseca R Greipp PR Dewald GW Kurtin PJ The (11;14)(q13;q32) translocation in multiple myeloma. A morphologic and immunohistochemical study.Am J Clin Pathol. 2000; 113: 831-837Crossref PubMed Scopus (109) Google Scholar, 15Vasef MA Medeiros LJ Yospur LS Sun NC McCourty A Brynes RK Cyclin D1 protein in multiple myeloma and plasmacytoma: an immunohistochemical study using fixed, paraffin-embedded tissue sections.Mod Pathol. 1997; 10: 927-932PubMed Google Scholar The reason(s) for cyclin D1 dysregulation in these latter cases are unknown. Because cyclin D1 is one of the main target genes of Stat 3, we wished to study the possible interaction of activated Stat 3 and cyclin D1 dysregulation in MM. For these reasons we analyzed in a series of primary MM: 1) the frequency of constitutively activated Stat 3, 2) the level of cyclin D1 mRNA and protein expression, and 3) the downstream effects of activated Stat 3 on proliferation and induced anti-apoptosis. Forty-eight formalin-fixed, paraffin-embedded and nondecalcified blocks of tissue specimens obtained from lytic bone lesions of MM diagnosed between 1991 and 2001 were selected from the files of the Institute of Pathology, Technical University of Munich, Munich, Germany. Most of the material was obtained during surgery, and contained a high percentage of tumor cells with few bone trabeculae. Clinical information was obtained from the patients' medical records. Hematoxylin and eosin-stained slides and immunoperoxidase studies were reviewed in all cases by three of the authors (LQ-M, MK, and FF). The cases were graded according to the histological grading criteria described by Bartl and colleagues17Bartl R Frisch B Fateh-Moghadam A Kettner G Jaeger K Sommerfeld W Histologic classification and staging of multiple myeloma: a retrospective and prospective study of 674 cases.Am J Clin Pathol. 1987; 87: 342-355Crossref PubMed Scopus (248) Google Scholar The clinical staging was done according to Durie and Salmon.18Durie BGM Salmon S A clinical staging system for multiple myeloma.Cancer. 1975; 36: 842-854Crossref PubMed Scopus (2617) Google Scholar Some of the cases have been reported previously as part of another study.19Specht K Kremer M Müller U Dirnhofer S Rosemann M Höfler H Quintanilla-Martinez L Fend F Identification of cyclin D1 mRNA overexpression in B-cell neoplasias by real-time RT-PCR of microdissected paraffin sections.Clin Cancer Res. 2002; 8: 2902-2911PubMed Google Scholar Immunohistochemistry was performed on an automated immunostainer (Ventana Medical Systems, Inc., Tucson, AZ) according to the company's protocols, with slight modifications. After deparaffinization and rehydration, the slides were placed in a microwave pressure cooker in 0.01 mol/L citrate buffer, pH 6.0, containing 0.1% Tween 20, and heated in a microwave oven at maximum power for 30 minutes. The antibody panel used included cyclin D1 (clone P2D11F11; Novocastra, Newcastle, UK), Stat3 (BD Transduction Laboratories; San Diego, CA), Stat3 phosphorylated (P) (New England Biolabs Inc., Beverly, MA), p21 (BD Transduction Laboratories), Bcl-2 (DAKO, Glostrup, Denmark), Bcl-xL (BD Transduction Laboratories), Mcl-1 (Chemicon, Temecula, CA), CD20 (clone L26, DAKO), and p27KIP1 (BD Transduction Laboratories). The proliferation rate was assessed with the monoclonal antibody against the Ki67 antigen (clone MiB-1, DAKO). Appropriate positive controls were used to confirm the adequacy of the staining. The Stat 3 antibody from Transduction Laboratories recognizes both the latent (cytoplasmic) and the activated (nuclear) form of Stat 3, whereas the Stat 3P antibody recognizes exclusively the nuclear or activated form of Stat 3. As control for the anti-Stat 3 antibodies, cell blocks from two MM cell lines were used. To assure the staining quality of cyclin D1, a cyclin D1-positive mantle cell lymphoma carrying the t(11;14) translocation was included in every run. Because p21 expression is found only in isolated lymphocytes, any staining >10% was considered positive. A grid ocular objective was used to count 300 cells over three high-power fields (×40) and the percentage of positive cells was reported as 0 to 100%. In addition to the primary cases, eight human myeloma cell lines (KMM1, OPM2, U266, KMS5, KMS11, KMS12, KMS18, KMS20) were used in this study. A mantle cell lymphoma cell line, Granta 519, was used as control. All cell lines were cultured in RPMI 1640 medium supplemented with 15% fetal calf serum, 2 mmol/L glutamine, 10 U/ml penicillin, and streptomycin (Gibco, Life Technologies). Protein concentration was determined by the BCA protein assay reagent kit (Pierce, Rockford, IL). Tumor cell lines were grown in RPMI 1640 supplemented with 15% fetal calf serum. The cells were harvested during their exponential growth phase, centrifuged at 550 × g for 5 minutes, and the supernatant removed. The cell pellet was washed once and resuspended in three drops of human plasma to which three drops of thrombin were added. The resulting clot was fixed in 10% formalin and paraffin-embedded. A total of 60 μg of protein extracts was separated by 7.5% and/or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to pure nitrocellulose immobilization membranes (Amersham Pharmacia Biotech, Freiburg, Germany). Membranes were blocked for 1 hour in 5% nonfat dry milk and incubated with the primary antibody for 1 hour. Subsequently, membranes were washed five times for 5 minutes each in a wash buffer (10 mmol/L Tris, pH 7.6, 100 mmol/L/L NaCl, and 0.1% Tween) and incubated with a biotinylated secondary antibody for 1 hour. Membranes were washed five times in the same wash buffer and detection was performed by chemiluminescence with the ECL detection system (Amersham Pharmacia Biotech) for 2 to 15 minutes, and then the membranes were exposed to a Kodak X-OMAT AR film. All assays were repeated several times and gave similar results. IL-6 was measured in the supernatant of the cell cultures, 5 days from splitting during their exponential growth phase using the Quantikine human IL-6 kit from R&D Systems (Minneapolis, MN). The enzyme-linked immunosorbent assay was performed according to the recommendations of the manufacturer without modifications. The optical density was measured with the microplate reader DigiScan from Asys Hithec, Germany. The used wavelength was 450 nm with a wavelength correction at 570 nm as recommended. The standard curve was created by reducing the data using a computer software (MiKrotek Labor Systems GmbH, Overath, Germany) capable to generate a four-parameter logistic (4-PL) curve fit as recommended. Tissue preparation, microdissection of pure tumor cell populations, and RNA extraction from formalin-fixed tissues for real-time quantitative RT-PCR were performed as described previously.20Specht K Richter T Müller U Walch A Werner M Höfler H Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue.Am J Pathol. 2001; 158: 419-429Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar Real-time RT-PCR reactions were performed using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Intron-spanning primers and probes for cyclin D1, Stat 3, and TATA box-binding protein (TBP) as housekeeping gene control were designed using Primer Express software (Applied Biosystems) (Table 1).Table 1Sequence of TaqMan Primers and Probes Used in This StudyOligonucleotideLocationSequenceSize PCR productStat 3 FP2284 FGCC AGA GAG CCA GGA GCAStat 3 RP2358 RACA CAG ATA AAC TTG GTC TTC AGG TAT GStat 3 probe2305 TTGA AGC TGA CCC AGG TAG CGC TGC74 bpCycD1 FP307 F5′-CCGTCCATGCGGAAGATC-3′CycD1 RP376 R5′-CCTCCTCCTCGCACTTCTGT-3′CycD1 probe331 T5′-CTCGCAGACCTCCAGCATCCAGGT-3′69 bpTBP FP645 F5′-GCCCGAAACGCCGAATAT-3′TBP RP717 R5′-CCGTGGTTCGTGGCTCTCT-3′TBP probe664 T5′-ATCCCAAGCGGTTTGCTGCGG-3′72 bp Open table in a new tab Ten μl of RNA extracted from microdissected cells was transcribed into cDNA using Superscript II reverse transcriptase (Invitrogen) and 250 ng of random hexamers (Roche, Penzberg, Germany) following the manufacturer's recommendations in a final volume of 20 μl. PCR reactions were performed in at least two replicates and were performed with the TaqMan Universal PCR Master Mix (Applied Biosystems) using 6 μl of diluted cDNA, 200 nmol/L of the labeled probe, and 300 nmol/L of primers (except cyclin D1-307 sense that was used at 900 nmol/L) in a 30-μl final reaction mixture. After initial incubation at 50°C for 2 minutes and 95°C for 10 minutes, samples were amplified for 50 cycles of 95°C for 15 seconds, followed by 60°C for 1 minute. Amounts of cyclin D1, Stat 3, and TBP mRNAs were calculated using linear regression analysis from an external standard curve generated from Granta 519 RNA. To determine the threshold value for altered cyclin D1 and Stat 3 expression in MM, the mean values of cyclin D1/TBP and Stat 3/TBP ratios in reactive tissues from lymph nodes and bone marrow were evaluated. The mean cyclin D1/TBP ratio in reactive tissues was 0.84 (range, 0.31 to 1.38). Cyclin D1/TBP ratios greater than the value of 2.33 (mean + 5 SD) were arbitrarily considered to represent increased cyclin D1 expression. The Stat3/TBP ratio in reactive tissues from BM and LN was also determined (mean, 1.27; range, 1.15 to 1.38). Stat3/TBP ratios below 1.79 (mean + 5 SD) were arbitrarily considered to represent normal Stat3 levels. The significance of the association of different clinicopathological parameters with the expression of Stat 3 and cyclin D1 or with the lack of expression of both proteins was assessed with the chi-square test. The association between Bcl-2 and proliferation rate was assessed using the Mann-Whitney U-test and the Fisher's exact test. The association between the expression of Stat 3P and cyclin D1 was assessed using the Fisher's exact test. Clinical data and histology are summarized in Table 2. Of the 48 patients, 25 were male and 23 were female (M:F ratio, 1:1), with a median age of 64 years (range, 32 to 81 years). In 12 patients the clinical information was not available. Histologically, 39 cases (81%) were composed of well-differentiated plasma cells (Bartl grade I) and 9 cases (19%) were moderately differentiated (Bartl grade II). Ten cases revealed lymphoplasmacytoid differentiation. None of the cases showed plasmablastic morphology.Table 2Clinicopathological Features of 48 MM Patients Included in the StudyCaseAgeSexMGHistologyStageTreatmentFollow-upGroup 1. Stat3P+156F***data are not available;I-LPIaRTDwD28 months269MG,λIIaCTANED26 months374FκIIIIa***data are not available;DwD28 months475FκIIIIaRT + CTDwD48 months564FG,λIIIIaRT + CTAwD30 months652MG,λIIaRTAwD22 months764MG,κIIIIIaRTDwD83 months879F***data are not available;I***data are not available;***data are not available;LFU954MλI-LPIIIaCTANED26 months1063MG,λIIaRT + CTDwD84 months1172F***data are not available;IIIa***data are not available;LFU1275M***data are not available;II***data are not available;***data are not available;LFU13*only the biopsy at relapse was available; T, autologous stem-cell bone marrow transplant; LP, lymphoplasmocytoid morphology.63FA,λIIIIaRT + CTAwR96 months1464MG,λIIIIaCTLFU1555FλIIIIaRT + CTAwD4 months1674FG,κIIIIaRT + CTAwD6 months1757M***data are not available;I***data are not available;***data are not available;LFU1867MG,κI-LPIaAwD36 months1932MG,κIIaRT + CT + TAwD53 months2074FG,κIIIIaRT + CTDwD12 months2162FM,λIIIaRTAwD12 months2260FG,λIIIIIaCTAwD22 months23*only the biopsy at relapse was available; T, autologous stem-cell bone marrow transplant; LP, lymphoplasmocytoid morphology.76MA,λIIIIIbRT + CTAwR51 monthsGroup 2. Cyclin D1 +2459M***data are not available;I-LP***data are not available;***data are not available;LFU2559MκI-LPIIIbCTDwD29 months2676MκI-LPIaRTAwD15 months2776FλIIIIaRT + CTDwD11 months2858MG,κI-LPIIIaRT + CTAwD87 months2956FG,κIIIIaRT + CTAwD91 months3078FG,κIIIIbRT + CTDwD48 months3164MG,κI-LPIa***data are not available;AwD1 month3281MλI-LPIIIaRT + CTDwD10 months3340MG,κIIIIbCT + TAwD36 months3461FκIIIIaRTAwD3 months3569FG,κIIaRTAwD25 months3678M***data are not available;I***data are not available;***data are not available;LFU3766MG,κI-LPIaCTAwD41 months3859MG,κIIaRTAwR75 monthsGroup 3. Stat3P- and cyclin D1-negative3973MG,κIIIIaRT + CTAwD54 months4076MG,κIIIIIa***data are not available;DwD23 months4163FG,κIIIIIaRTLFU4248FλIIIIIaRT + CT + TAwD46 months4369FG,κIIIIaRT + CTLFU4471FG,κIIIIbRT + CTAwD29 months4547FκIIIIaRT + CTDwD67 months4661M***data are not available;IIa***data are not available;LFU4760FG,κIIIIaRT + CTAwD27 months4868M***data are not available;I***data are not available;***data are not available;LFUMG, monoclonal gammopathy; AwD, alive with disease; DwD, died with disease; ANED, alive no evidence of disease; LFU, lost to follow-up; AwR, alive with recurrence; CT, chemotherapy; RT, radiotherapy;*** data are not available;* only the biopsy at relapse was available; T, autologous stem-cell bone marrow transplant; LP, lymphoplasmocytoid morphology. Open table in a new tab MG, monoclonal gammopathy; AwD, alive with disease; DwD, died with disease; ANED, alive no evidence of disease; LFU, lost to follow-up; AwR, alive with recurrence; CT, chemotherapy; RT, radiotherapy; To validate the specificity of the Stat 3 antibodies used in this study, Western blot analysis was performed in well-characterized human MM cell lines (Figure 1). Western blot analysis with the N-terminal-specific anti-Stat 3 antibody that recognizes both Stat 3α- and Stat 3β-phosphorylated and -unphosphorylated proteins, demonstrated two specific protein bands of the expected size (92 and 83 kd, respectively) in all human MM cell lines. The mantle cell lymphoma cell line, Granta 519, used as control for cyclin D1 expression showed significantly weaker bands. In contrast, phosphorylated Stat 3 (Y705) (92 kd), which specifically recognizes Stat 3 phosphorylated at tyrosine 705, was strongly expressed only in U266 and KMS20 MM cell lines. As expected, cyclin D1 was expressed only in the two cell lines known to have a t(11;14); Granta 519 and the MM cell line KMS12. Bcl-xL and Mcl-1 were expressed in all cell lines and their levels of expression varied from one cell line to another (Figure 1). Five MM cell lines expressed high levels of Bcl-xL (U266, KMS18, KMS5, KMS11, and KMM1), and three expressed moderate levels (KMS20, KMS12, and OPM2). The expression of Mcl-1 was more homogeneous and only U266 expressed higher levels when compared to the other cell lines. Bcl-2 was negative in three of the eight cell lines (KMS-20, KMS-5, and KMM1). Of note is that the two cell lines with t(11;14) and expression of cyclin D1 showed very high levels of Bcl-2. IL-6 was measured in the supernatant of all cell lines to identify those MM cell lines with autocrine production of IL-6. We found that KMS-20, U266, KMS18, KMS5, and KMM1 produce IL-6 in an autocrine manner, whereas the mantle cell lymphoma cell line Granta 519 and the MM cell lines KMS11, KMS12, and OPM2 are independent of IL-6 for their growth. Of note is that even though all cell lines expressed Stat 3 in steady-state, those cell lines with autocrine production of IL-6 had higher expression levels of Stat 3, with the exception of KMS18. However, the autocrine production of IL-6 in some cell lines did not universally result in the presence of Stat 3P, nor did it influence the expression of Bcl-xL, Mcl-1, and Bcl-2. The results of the immunohistochemical studies are summarized in Table 3. All cases were previously immunophenotyped for CD20 and CD138. All cases were positive for CD138. Four of the 48 cases (8.3%) showed expression of CD20 in a proportion of the tumor cells. To confirm the staining pattern of the Stat 3 antibodies in paraffin tissues, cell blocks from two MM cell lines were used. The KMS18 cell line showed a purely cytoplasmic staining with the Stat 3 antibody demonstrating the presence of steady-state Stat 3 (Figure 2A) and was negative for the Stat 3P antibody (Figure 2A, inset). In contrast the U266 cell line reveale