Retinoblastoma Protein Is Frequently Absent or Phosphorylated in Anaplastic Large-Cell Lymphoma
2004; Elsevier BV; Volume: 164; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)63782-0
ISSN1525-2191
AutoresGeorge Z. Rassidakis, Raymond Lai, Marco Herling, Candy C. Cromwell, Annette Schmitt‐Graeff, L. Jeffrey Medeiros,
Tópico(s)Cancer-related Molecular Pathways
ResumoThe possible role of retinoblastoma protein (Rb) in the pathogenesis of anaplastic large-cell lymphoma (ALCL) is unknown. We investigated Rb protein expression, both total (phosphorylated and underphosphorylated) and active (underphosphorylated), in four anaplastic lymphoma kinase (ALK)-positive ALCL cell lines (Karpas 299, JB-6, SU-DHL1, and SR-786) by Western blot analysis, and in 67 ALCL tumors (30 ALK-positive, 37 ALK-negative) using immunohistochemical methods. We also used fluorescence in situ hybridization and polymerase chain reaction methods to assess for loss of heterozygosity of the rb gene. The findings were correlated with apoptotic rate assessed by the terminal dUTP nick-end labeling assay. Immunoblots showed high total Rb levels in Karpas 299, SU-DHL1 and SR-786 and relatively lower levels in and JB-6. Underphosphorylated Rb was negative or expressed at low levels in all cell lines. In ALCL tumors, total Rb was detected in 44 (66%) and absent in 23 (34%). The mean apoptotic rate was 3.2% in Rb-negative tumors compared with 2.7%, 2.2%, and 1.2% in tumors with 50% Rb-positive cells, respectively (P = 0.2, Kruskall-Wallis test). In a subset of 25 total Rb-positive tumors we assessed for underphosphorylated Rb, which was detected in 12 tumors. The detection of only total Rb in the remaining 13 tumors suggests that Rb was phosphorylated. Fluorescence in situ hybridization showed allelic loss of the rb gene in 10 (40%) of 25 tumors analyzed and was significantly associated with absence of Rb expression (P = 0.003). Similar results were obtained for loss of heterozygosity of the 13q14 locus. Five-year progression-free survival for patients with Rb-negative ALCL was 89.4% compared with 47.7% for patients with total Rb-positive ALCL (P = 0.006, log-rank test). Similar trends for progression-free survival held true for patients with ALK-positive and ALK-negative tumors analyzed separately. In conclusion, Rb is absent or phosphorylated in most ALCL cell lines and tumors and absence of Rb expression is associated with better clinical outcome in patients with ALCL. The possible role of retinoblastoma protein (Rb) in the pathogenesis of anaplastic large-cell lymphoma (ALCL) is unknown. We investigated Rb protein expression, both total (phosphorylated and underphosphorylated) and active (underphosphorylated), in four anaplastic lymphoma kinase (ALK)-positive ALCL cell lines (Karpas 299, JB-6, SU-DHL1, and SR-786) by Western blot analysis, and in 67 ALCL tumors (30 ALK-positive, 37 ALK-negative) using immunohistochemical methods. We also used fluorescence in situ hybridization and polymerase chain reaction methods to assess for loss of heterozygosity of the rb gene. The findings were correlated with apoptotic rate assessed by the terminal dUTP nick-end labeling assay. Immunoblots showed high total Rb levels in Karpas 299, SU-DHL1 and SR-786 and relatively lower levels in and JB-6. Underphosphorylated Rb was negative or expressed at low levels in all cell lines. In ALCL tumors, total Rb was detected in 44 (66%) and absent in 23 (34%). The mean apoptotic rate was 3.2% in Rb-negative tumors compared with 2.7%, 2.2%, and 1.2% in tumors with 50% Rb-positive cells, respectively (P = 0.2, Kruskall-Wallis test). In a subset of 25 total Rb-positive tumors we assessed for underphosphorylated Rb, which was detected in 12 tumors. The detection of only total Rb in the remaining 13 tumors suggests that Rb was phosphorylated. Fluorescence in situ hybridization showed allelic loss of the rb gene in 10 (40%) of 25 tumors analyzed and was significantly associated with absence of Rb expression (P = 0.003). Similar results were obtained for loss of heterozygosity of the 13q14 locus. Five-year progression-free survival for patients with Rb-negative ALCL was 89.4% compared with 47.7% for patients with total Rb-positive ALCL (P = 0.006, log-rank test). Similar trends for progression-free survival held true for patients with ALK-positive and ALK-negative tumors analyzed separately. In conclusion, Rb is absent or phosphorylated in most ALCL cell lines and tumors and absence of Rb expression is associated with better clinical outcome in patients with ALCL. The retinoblastoma (rb) gene, located at 13q14, was the first tumor suppressor gene identified by its involvement in hereditary retinoblastoma.1Lee WH Bookstein R Hong F Young LJ Shew JY Lee EY Human retinoblastoma susceptibility gene: cloning, identification, and sequence.Science. 1987; 235: 1394-1399Crossref PubMed Scopus (1065) Google Scholar, 2Fung YK Murphree AL T'Ang A Qian J Hinrichs SH Benedict WF Structural evidence for the authenticity of the human retinoblastoma gene.Science. 1987; 236: 1657-1661Crossref PubMed Scopus (584) Google Scholar, 3Knudson Jr, AG Retinoblastoma: a prototypic hereditary neoplasm.Semin Oncol. 1978; 5: 57-60PubMed Google Scholar Accumulating evidence from in vitro and in vivo studies suggests that the rb gene product, a 105-kd protein, is implicated in many cellular functions including cell proliferation, apoptosis, and differentiation.4Classon M Harlow E The retinoblastoma tumour suppressor in development and cancer.Nat Rev Cancer. 2002; 2: 910-917Crossref PubMed Scopus (596) Google Scholar The role of Rb protein as a major cell-cycle inhibitor is mediated through its interaction with the E2F transcription factors.5Helin K Lees JA Vidal M Dyson N Harlow E Fattaey A A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F.Cell. 1992; 70: 337-350Abstract Full Text PDF PubMed Scopus (522) Google Scholar, 6Kaelin Jr, WG Krek W Sellers WR DeCaprio JA Ajchenbaum F Fuchs CS Chittenden T Li Y Farnham PJ Blanar MA Livingston DM Flemington EK Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties.Cell. 1992; 70: 351-364Abstract Full Text PDF PubMed Scopus (692) Google Scholar Rb directly binds and inhibits the transcriptional activity of E2F family members by recruitment of several chromatin-remodeling complexes to promoter regions, resulting in chromatin condensation and inhibition of transcription.7Chellappan SP Hiebert S Mudryj M Horowitz JM Nevins JR The E2F transcription factor is a cellular target for the RB protein.Cell. 1991; 65: 1053-1061Abstract Full Text PDF PubMed Scopus (1093) Google Scholar, 8Johnson DG Schwarz JK Cress WD Nevins JR Expression of transcription factor E2F1 induces quiescent cells to enter S phase.Nature. 1993; 365: 349-352Crossref PubMed Scopus (833) Google Scholar, 9Harbour JW Dean DC Corepressors and retinoblastoma protein function.Curr Top Microbiol Immunol. 2001; 254: 137-144PubMed Google Scholar As a result, cells undergo arrest in the G1 phase of the cell cycle.10Sherr CJ Cancer cell cycles.Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4945) Google Scholar Although Rb is normally expressed throughout the entire cell cycle, its function depends on its phosphorylation status.11Goodrich DW Wang NP Qian YW Lee EY Lee WH The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle.Cell. 1991; 67: 293-302Abstract Full Text PDF PubMed Scopus (613) Google Scholar Underphosphorylated Rb is the active, growth suppressive form of the protein, and phosphorylated Rb is inactive, incapable of binding to E2F proteins. Progressive phosphorylation of Rb in mid to late G1 phase of the cell cycle, by cyclin/cyclin-dependent kinase (CDK) complexes, results in release of E2F proteins, transcription of numerous target genes, and entry of the cell cycle into S phase. CDKs, in turn, are negatively regulated by their inhibitors including the INK4 (p14ARF, p15, p16, p18) and Cip/Kip (p21, p27, p57) families.12Sherr CJ Roberts JM CDK inhibitors: positive and negative regulators of G1-phase progression.Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5097) Google Scholar Rb is also involved in apoptotic mechanisms. 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France, IARC Press, Lyon2001: 230-235Google Scholar, 26Morris SW Kirstein MN Valentine MB Dittmer KG Shapiro DN Saltman DL Look AT Fusion of a kinase gene ALK, to a nucleolar protein gene NPM, in non-Hodgkin's lymphoma.Science. 1994; 263: 1281-1284Crossref PubMed Scopus (1936) Google Scholar, 27Morris SW Naeve C Mathew P James PL Kirstein MN Cui X Witte DP ALK, the chromosome 2 gene locus altered by the t(2;5) in non-Hodgkin's lymphoma, encodes a novel neural receptor tyrosine kinase that is highly related to leukocyte tyrosine kinase (LTK).Oncogene. 1997; 14: 2175-2188Crossref PubMed Scopus (409) Google Scholar We hypothesized that the Rb pathway may be altered in ALCL because previous studies of these tumors have shown deregulation of other cell-cycle-controlling proteins.28Inghirami G Macri L Cesarman E Chadburn A Zhong J Knowles DM Molecular characterization of CD30+ anaplastic large-cell lymphoma: high frequency of c-myc proto-oncogene activation.Blood. 1994; 83: 3581-3590PubMed Google Scholar, 29Chilosi M Doglioni C Magalini A Inghirami G Krampera M Nadali G Rahal D Pedron S Benedetti A Scardoni M Macri E Lestani M Menestrina F Pizzolo G Scarpa A p21/WAF1 cyclin-kinase inhibitor expression in non-Hodgkin's lymphomas: a potential marker of p53 tumor-suppressor gene function.Blood. 1996; 88: 4012-4020PubMed Google Scholar, 30Chilosi M Doglioni C Yan Z Lestani M Menestrina F Sorio C Benedetti A Vinante F Pizzolo G Inghirami G Differential expression of cyclin-dependent kinase 6 in cortical thymocytes and T-cell lymphoblastic lymphoma/leukemia.Am J Pathol. 1998; 152: 209-217PubMed Google Scholar, 31Rassidakis GZ Claret FX Lai R Zhang Q Sarris AH McDonnell TJ Medeiros LJ Expression of p27(Kip1) and c-Jun activation binding protein 1 are inversely correlated in systemic anaplastic large cell lymphoma.Clin Cancer Res. 2003; 9: 1121-1128PubMed Google Scholar To investigate the functional status of Rb in ALCL, we assessed a subset of these tumors for expression of underphosphorylated Rb. We also used fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) methods to assess for possible loss of rb alleles. We correlated these results with tumor apoptotic rate (AR), proliferation index, and clinical outcome.Materials and MethodsCell Lines and Western Blot AnalysisFour ALK-positive ALCL cell lines, all known to carry the t(2;5) were used, including Karpas 299 (a gift from Dr. M. Kadin, Beth-Israel-Deaconess Medical Center, Boston, MA), SR-786, SU-DHL-1 (both from DSMZ, Braunschweig, Germany), and JB-6 (a gift from Dr. D. Jones, M.D. Anderson Cancer Center, Houston, TX). The cell lines were maintained in RPMI 1640 medium supplemented with 1% nonessential amino acids, 10% fetal calf serum (Invitrogen Corp., Grand Island, NY), and 1% streptomycin-penicillin. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. For the serum starvation experiments, Karpas 299 and SU-DHL-1 cells were maintained in RPMI 1640 medium supplemented with only 1% fetal calf serum (Invitrogen Corp.), and 1% streptomycin-penicillin. Lysates from serum-starved cells were prepared before the experiment (control or time point 0 hours) and after 48 and 72 hours.Total protein was extracted from these cell lines and Western blot analysis performed using methods described previously.31Rassidakis GZ Claret FX Lai R Zhang Q Sarris AH McDonnell TJ Medeiros LJ Expression of p27(Kip1) and c-Jun activation binding protein 1 are inversely correlated in systemic anaplastic large cell lymphoma.Clin Cancer Res. 2003; 9: 1121-1128PubMed Google Scholar Primary antibodies specific for total Rb (clone Rb1; DAKO, Carpinteria, CA) and underphosphorylated Rb (clone G99-549; BD Biosciences Pharmingen, San Diego, CA) were used. We further tested the specificity of the underphosphorylated Rb antibody using Western blot analysis and lysates of previously serum-starved Karpas 299 cells. Because the underphosphorylated Rb migrates faster in 6% polyacrylamide gels than the phosphorylated form of the protein (higher molecular weight), immunoblots using the underphosphorylated Rb antibody revealed the presence of a band corresponding only to a lower molecular weight (underphosphorylated) protein product. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Ambion, Austin, TX) was used as a control for protein load and integrity.ALCL TumorsThis group included 67 cases of systemic ALCL obtained from previously untreated patients accessioned at The University of Texas M.D. Anderson Cancer Center and the University of Freiburg, Germany from 1982 to 2001. The diagnosis of ALCL was based on morphological and immunohistological criteria as specified by the World Health Organization classification.25Delsol G Ralfkiaer E Stein H Wright D Jaffe ES Anaplastic large cell lymphoma.in: Jaffe ES Harris NL Stein H Vardiman JW Pathology and Genetics of Tumors of Haematopoietic and Lymphoid Tissues. France, IARC Press, Lyon2001: 230-235Google Scholar ALK-positive lymphomas, regardless of morphological features (ie, classical, monomorphous, lymphohistiocytic), were diagnosed as ALCL. ALK-negative ALCL tumors were composed predominantly of large anaplastic cells. Some investigators, as acknowledged in the World Health Organization classification, believe that ALK-negative ALCLs are better classified as high-grade peripheral T-cell lymphoma unspecified. All ALCL tumors were uniformly positive for CD30 and negative for B-cell antigens, including CD20, CD79a, and PAX-5. Fifty-three (79%) tumors were T cell and 14 (21%) were null cell. ALK was assessed using the ALK-1 antibody (1:30, DAKO) and was positive in 30 (45%) cases.All patients were treated with doxorubicin-based chemotherapy. The clinicopathological features of most of these patients have been reported previously.32Rassidakis GZ Sarris AH Herling M Ford RJ Cabanillas F McDonnell TJ Medeiros LJ Differential expression of BCL-2 family proteins in ALK-positive and ALK-negative anaplastic large cell lymphoma of T/null-cell lineage.Am J Pathol. 2001; 159: 527-535Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar The median age of patients with ALK-positive tumors was 29 years compared with 52 years for patients with ALK-negative tumors (P < 0.0001 by Mann-Whitney U-test). Patients with ALK-negative tumors were more frequently associated with anemia (P = 0.03, Fisher's exact test). All other clinicopathological parameters between the two groups were comparable.Design and Construction of the Tissue ArrayTissue sections of ALCL tumors, 5 μm thick, were cut from whole paraffin blocks (19 tumors) or a tissue microarray (48 tumors). The tissue microarray included triplicate or quadruplet tumor cores from all 48 tumors and two reactive lymph nodes and was constructed using a manual tissue arrayer (Beecher Instruments, Silver Springs, MD) as described previously.33Rassidakis GZ Jones D Thomaides A Sen F Lai R Cabanillas F McDonnell TJ Medeiros LJ Apoptotic rate in peripheral T-cell lymphomas: a study using a tissue microarray with validation on full tissue sections.Am J Clin Pathol. 2002; 118: 328-334Crossref PubMed Scopus (60) Google ScholarImmunohistochemical Methods and Terminal dUTP Nick-End Labeling (TUNEL) AssayThe immunohistochemical methods used in this study have been described previously.32Rassidakis GZ Sarris AH Herling M Ford RJ Cabanillas F McDonnell TJ Medeiros LJ Differential expression of BCL-2 family proteins in ALK-positive and ALK-negative anaplastic large cell lymphoma of T/null-cell lineage.Am J Pathol. 2001; 159: 527-535Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar For all antibodies, heat-induced epitope retrieval was performed. Rb was assessed using monoclonal antibodies specific for total Rb (clone Rb1, dilution 1:50; DAKO) and underphosphorylated Rb (clone G99-549, dilution 1:25; BD Biosciences). The latter antibody recognizes an epitope between amino acids 514 to 610 of human Rb and does not recognize the phosphorylated forms of the protein. Proliferation rate was assessed using the MIB-1 (Ki-67) antibody (dilution 1:120; Immunotech, Westbrook, ME). The slides were incubated with the total and underphosphorylated Rb monoclonal antibodies at 4°C overnight, and with MIB-1 at room temperature for 1 hour. Detection of the immunoreaction was performed using the LSAB+ kit (DAKO). Reactive small lymphocytes in all tissue sections served as internal positive controls for total Rb and underphosphorylated Rb. Slides stained with normal rabbit serum (DAKO) without primary antibody were used as negative controls.Any nuclear staining of ALCL cells was considered positive, irrespective of intensity. Expression levels for Rb were determined by counting the percentage of positive tumor cells and, for the purpose of statistical analysis, the tumors were divided into five groups as follows: negative, 90% positive. Proliferation index was designated as the percentage of MIB1-positive tumor nuclei. AR was assessed using a modified TUNEL assay and designated as the percentage of TUNEL-positive tumor nuclei as described elsewhere.32Rassidakis GZ Sarris AH Herling M Ford RJ Cabanillas F McDonnell TJ Medeiros LJ Differential expression of BCL-2 family proteins in ALK-positive and ALK-negative anaplastic large cell lymphoma of T/null-cell lineage.Am J Pathol. 2001; 159: 527-535Abstract Full Text Full Text PDF PubMed Scopus (93) Google ScholarFISHFISH was performed on a 5-μm paraffin section from the tissue microarray used in this study after appropriate deparaffinization in fresh xylene. Our FISH methods have been described in detail elsewhere.34Katz RL Caraway NP Gu J Jiang F Pasco-Miller LA Glassman AB Luthra R Hayes KJ Romaguera JE Cabanillas FF Medeiros LJ Detection of chromosome 11q13 breakpoints by interphase fluorescence in situ hybridization. A useful ancillary method for the diagnosis of mantle cell lymphoma.Am J Clin Pathol. 2000; 114: 248-257Crossref PubMed Scopus (46) Google Scholar, 35Jiang F Lin F Price R Gu J Medeiros LJ Zhang HZ Xie SS Caraway NP Katz RL Rapid detection of IgH/BCL2 rearrangement in follicular lymphoma by interphase fluorescence in situ hybridization with bacterial artificial chromosome probes.J Mol Diagn. 2002; 4: 144-149Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar We used the Spectrum Orange LSI 13/RB-1 and the CEP 8 probes purchased from Vysis (Downers Grove, IL) to assess the rb gene and the centromeric region (8p11.1-q11.1) of chromosome 8 (control), respectively. The tissue microarray slide was counterstained with 4,6-diamidino-2-phenylindole in an anti-fade mounting fluid before it completely dried. The signal was viewed at ×1000 magnification with a Zeiss fluorescence microscope (Carl Zeiss, Thornwood, NY) using Vysis FISH filters specific for 4,6-diamidino-2-phenylindole/Spectrum Orange. We analyzed at least 100 evaluable nuclei of each tumor. Only isolated cells with nonoverlapping nuclei were counted. To avoid overestimation of tumors with allelic loss of rb because of sectioning of the tumor cells, we defined allelic loss of rb as being present using a conservative cutoff of 50% or more cells that had only one signal with the RB1 probe. Most small reactive lymphocytes cut in full cross-section in tumor cores showed two signals and served as internal positive controls for the presence of both rb alleles.Loss of Heterozygosity (LOH) StudiesAfter histological examination of 37 ALCL tumors (12 ALK-positive, 25 ALK-negative), areas of tumor infiltration were delineated microscopically, and most of the uninvolved lymph node or other normal tissue in the paraffin blocks was removed with sterile surgical blades. Subsequently, 20-μm sections were cut from the trimmed blocks and genomic DNA was extracted using the QIAamp DNA extraction kit (Qiagen, Valencia, CA) according to the manufacturer's instructions.To determine LOH, genomic DNA was amplified using a PCR method and two microsatellite markers, D13S153 and D13S263, spanning the 13q14 locus. Both microsatellite markers are known to have a high frequency of heterozygosity (82% and 84%, respectively). The D13S153 and D13S263 markers were labeled with the fluorescent dyes TET and HEX, respectively, and were purchased from Research Genetics (Huntsville, AL). In addition, a control microsatellite marker derived from the 13q32 locus and labeled with HEX (Research Genetics) was used. The 50-μl reaction mixture contained 500 ng of DNA (5 μl), 5 μl of 10× PCR buffer (Invitrogen Corp.), 1.5 mmol/L MgCl2, 200 μmol/L each dNTP, 200 nmol/L each primer pair (Research Genetics) and 2.5 U of TaqDNA polymerase (Invitrogen Corp.). The TaqDNA polymerase was added to the reaction mixture after the denaturation step (hot start). The thermal cycler was programmed as follows: 95°C for 10 minutes (denaturation); 30 cycles of 45 seconds at 94°C, 45 seconds at 54°C, and 60 seconds at 72°C; and finally 10 minutes at 72°C. The presence of the PCR products was confirmed by electrophoresis on a 1.5% agarose gel. Amplified products were subsequently analyzed by high-resolution capillary electrophoresis using the 310-Genetic Analyzer (PE/Applied Biosystems, Foster City, CA), as previously described.36Vega F Medeiros LJ Jones D Abruzzo LV Lai R Manning J Dunmire V Luthra R A novel four-color PCR assay to assess T-cell receptor gamma gene rearrangements in lymphoproliferative lesions.Am J Clin Pathol. 2001; 116: 17-24Crossref PubMed Scopus (93) Google Scholar Fluorescence data were analyzed using GeneScan and Genotyper software (PE/Applied Biosystems). Informative cases were heterozygous for the RB1 locus. Cases homozygous for this locus were considered unevaluable for LOH. In heterozygous cases, a decrease in signal intensity of more than 50% between the two alleles was required to establish the presence of LOH.Statistical AnalysisThe chi-square and Fisher's exact tests were used to compare total and underphosphorylated Rb expression (positive versus negative) with various clinicopathological parameters. The Mann-Whitney U-test and the Kruskal-Wallis test were chosen for the nonparametric correlation of proliferation index and AR between various Rb expression levels. Progression-free survival (PFS) was defined as time from initiation of therapy to last clinical follow-up, primary treatment failure, or relapse. Univariate survival analysis was based on the method of Kaplan and Meier using the log-rank test. Multivariate analysis was performed using the Cox proportional hazards model. All computations were performed using the StatView statistical program (Abacus Concepts, Inc., Berkeley, CA).ResultsCell LinesTotal Rb was expressed at high levels in Karpas 299, SU-DHL1, and SR-786 cells and at lower levels in JB-6 cells (Figure 1A). Immunoblots showed a thick band in positive cell lines, consistent with phosphorylated and hyperphosphorylated forms of Rb.19Galteland E Smedshammer L Suo Z DeAngelis P Stokke T Proliferation-dependent expression and phosphorylation of pRB in B cell non-Hodgkin's lymphomas: dependence on RB1 copy number.Leukemia. 2002; 16: 1549-1555Crossref PubMed Scopus (6) Google Scholar Underphosphorylated Rb was detected at low levels in all four ALCL cell lines (Figure 1A). Serum starvation of SU-DHL1 and Karpas 299 cells resulted in an increased underphosphorylated Rb/total Rb ratio indicating that a subset of these cells underwent cell-cycle arrest through activation of Rb protein (Figure 1B).Expression of Total Rb in ALCL TumorsIn reactive lymph nodes, total Rb was detected predominantly in germinal center cells as described previously.37Martinez JC Piris MA Sanchez-Beato M Villuendas R Orradre JL Algara P Sanchez-Verde L Martinez P Retinoblastoma (Rb) gene product expression in lymphomas. Correlation with Ki67 growth fraction.J Pathol. 1993; 169: 405-412Crossref PubMed Scopus (51) Google Scholar Total Rb was detected in 44 (66%) ALCL, including 18 of 30 (60%) ALK-positive and 26 of 37 (70%) ALK-negative tumors (P = 0.4, Fisher's exact test). Rb immunoreactivity was localized in the tumor nuclei with variable staining intensity (Figure 2). Three tumors had >90% cells positive, 11 tumors had >50 to 90% cells positive, 15 tumors had 10 to 50% cells positive, and 15 tumors had <10% cells positive. Twenty-three (34%) ALCLs showed complete absence of Rb (Figure 2) including 12 ALK-positive and 11 ALK-negative. Rb expression in ALCL did not significantly correlate with clinical and laboratory features in these patients.Figure 2Total Rb expression in ALCL tumors. A: A Rb-positive ALCL case with strong nuclear immunoreactivity in most tumo
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