|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, 1 April 2007, Vol. 109, No. 7, pp. 3060-3068. Prepublished online as a Blood First Edition Paper on November 30, 2006; DOI 10.1182/blood-2006-07-036368.
NEOPLASIA IRF-4 and c-Rel expression in antiviral-resistant adult T-cell leukemia/lymphoma1 Division of Hematology/Oncology, Department of Medicine, 2 Department of Pathology, 4 Division of Biostatics, Department of Epidemiology and Public Health, and 6 Department of Microbiology and Immunology, University of Miami Miller School of Medicine, FL; 3 Division of Oncology, Department of Medicine, Washington University, St Louis, MO; 5 Division of Infectious Disease, Department of Medicine, Federal University of Bahia, Salvador, Brazil
Adult T-cell leukemia/lymphoma (ATLL) is a generally fatal malignancy. Most ATLL patients fare poorly with conventional chemotherapy; however, antiviral therapy with zidovudine (AZT) and interferon alpha (IFN-  ) has produced long-term clinical remissions. We studied primary ATLL tumors and identified molecular features linked to sensitivity and resistance to antiviral therapy. Enhanced expression of the proto-oncogene c-Rel was noted in 9 of 27 tumors. Resistant tumors exhibited c-Rel (6 of 10; 60%) more often than did sensitive variants (1 of 9; 11%). This finding was independent of the disease form. Elevated expression of the putative c-Rel target, interferon regulatory factor-4 (IRF-4), was observed in 10 (91%) of 11 nonresponders and in all tested patients with c-Rel+ tumors and occurred in the absence of the HTLV-1 oncoprotein Tax. In contrast, tumors in complete responders did not express c-Rel or IRF-4. Gene rearrangement studies demonstrated the persistence of circulating T-cell clones in long-term survivors maintained on antiviral therapy. The expression of nuclear c-Rel and IRF-4 occurs in the absence of Tax in primary ATLL and is associated with antiviral resistance. These molecular features may help guide treatment. AZT and IFN- is a suppressive rather than a curative regimen, and patients in clinical remission should remain on maintenance therapy indefinitely.
Adult T-cell leukemia/lymphoma (ATLL) was first described as a distinct clinical entity in 1979, and the association with the human T-cell leukemia virus type 1 (HTLV-1) was reported shortly thereafter.1 The disease may manifest itself in various forms and is generally subclassified into 4 subtypes.2 In the 2 most aggressive variants, lymphoma-type and acute ATLL, patients usually have a very high tumor burden and hypercalcemia. The chronic and smoldering variants of ATLL have a more indolent course, though they often progress to the more malignant forms of the disease.3 Therapy for ATLL, particularly acute and lymphoma types, is disappointing. In a large published series of more than 800 Japanese patients with acute and lymphomatous ATLL who were treated with a variety of chemotherapeutic regimens, the median survival time was 6.2 and 10.2 months, respectively.2 With some of the most intensive chemotherapy regimens, complete response (CR) rates of approximately 35% or more have been reported.4,5 Allogeneic bone marrow transplantation, including reduced-intensity regimens, has been successful in a number of ATLL patients, though severe immunodeficiency resulting from the underlying disease and the preparatory regimens poses a significant problem.6,7 IL-2 receptor–directed therapies (anti-Tac) have proven to be useful in some ATLL patients,8,9 but these are also expensive and unlikely to be feasible in many areas in which HTLV-1 is endemic.
Several phase 2 trials have demonstrated the efficacy of zidovudine (AZT) and interferon alpha (IFN-
The study of the evolution of ATLL is further complicated by its low incidence (2%-6% lifetime risk) and prolonged latency (more than 30 years) before the development of overt disease in HTLV-1 carriers.20 In addition, the difficulty of establishing representative animal models and primary tumor cell lines has hindered research. In general, published "ATLL" cell lines are either clonal outgrowths that differ from the original tumor or HTLV-1–transformed cells that express the viral oncoprotein Tax.21 Most research on the pathogenesis of HTLV-1–related disease has focused on Tax, a promiscuous transcriptional activator that induces the expression of viral genes (through the viral LTR) and cellular genes through interaction with pleiotropic transcription factors such as NF-
We studied and characterized the expression of activated NF-
Patients and tumor samples We studied a primary ATLL tumor cohort of 38 patients, mostly Caribbean immigrants from Miami, whose disease was diagnosed and treated between 1995 and 2006. Investigators at the Federal University of Bahia in the northeastern Brazilian state of Bahia, a highly endemic region for HTLV-1, also contributed tumor specimens for this study. ATLL was classified into clinical subtypes according to Shimoyama.2 The diagnosis of ATLL was based on serologic evidence of HTLV-1 by ELISA, confirmed by Western blot, and the identification of clonal circulating leukemic cells or tissue lymphocytes of T-cell origin was determined by flow cytometry, immunostains, and gene rearrangement studies. Patients also had one or more of the typical clinical features of the disease, including markedly elevated lactate dehydrogenase (LDH), generalized lymphadenopathy, hepatosplenomegaly, and hypercalcemia. Patient characteristics included in this study are summarized in Table 1.
The study was reviewed and approved by the institutional review boards of the University of Miami and the Federal University of Bahia. Primary ATLL specimens used for molecular studies were obtained from peripheral blood mononuclear cells (PBMCs) from leukemic patients or residual solid tumor tissue from patients with ATLL after informed consent was obtained. Specimens were either processed immediately for protein and nucleic acid extractions or cryopreserved for later use. Also included in this study were cryopreserved tumor samples from deceased patients. Twenty-one samples were derived from acute leukemia-type ATLL, and the other specimens were from chronic (1), unfavorable chronic (3), and lymphoma-type leukemia (13) ATLL (Table 1). In most patients, all tumor characterization studies were performed on the original diagnostic specimens before any treatment. Leukemic ATLL was found in 25 treatment-naive patients. Two patients, ATLL-1 and ATLL-11, initially were given diagnoses of T-cell lymphoma. Both had relapses with leukemia-type ATLL after receiving chemotherapy, at which time their PBMCs were preserved. Conventional chemotherapy failed in 4 patients (ATLL-6, ATLL-12, ATLL-20, ATLL-23), and their original diagnostic specimens were analyzed. ATLL-20 initially had lymphoma and experienced relapse with leukemia, and 5 patients (ATLL-5, ATLL-10, ATLL-32, ATLL-35, ATLL-37) were coinfected with HIV.
Thirty-eight samples were available for our study. Twenty-two patients treated in the same manner with antiviral therapy had evaluable responses. Twenty-seven of 38 samples were evaluated for expression of NF- Antiviral therapy and response criteria
Twenty-eight patients in this study (21 of whom were treatment naive) received high-dose parenteral AZT (1.5 g twice daily) and IFN- Multiplex polymerase chain reaction Multiplex polymerase chain reaction (PCR) analysis for T-cell receptor (TCR) gene rearrangement (performed with reagents from Invivoscribe Technologies, San Diego, CA) were carried out as routine diagnostic work-up for lymphoma, as previously described.32,33 Briefly, purified DNA was quantified and amplified using a thermal cycler (ABI Geneamp 9700; Applied Biosystems, Foster City, CA). Detection was performed using an ABI 3100-Avant Genetic Analyzer (Applied Biosystems). Fragment analysis using GeneMapper version 3.7 software was used to detect the fluorescence-labeled amplified products. DNA from normal polyclonal populations gave a unique Gaussian distribution of amplified products in the expected size range for the primers used. Monoclonal populations gave unique, single-sized peaks, with or without a significant polyclonal background. Antibodies and reagents
The following antibodies were used for electrophoretic mobility shift assay (EMSA) supershift experiments: anti-p50 (H-119), anti-p65 (F-6), anti-c-Rel (N), p52 (C-5), and RelB (C-19) (Santa Cruz Biotechnology, Santa Cruz, CA). For Western blot analysis, the following antibodies were used: anti–IRF-4 (Santa Cruz Biotechnology), mouse monoclonal anti-Tax from hybridoma cell culture (kindly provided by Dr Chou-Zen Giam), anti–ß-actin, and anti– Isolation of PBMCs from ATLL patients and cell culture PBMCs from ATLL patients with leukemia, an HTLV-1–negative patient with peripheral CD4+CD25+ T-cell lymphoma with leukemia phase, and a healthy donor were separated using Lymphoprep solution (MediaTech, Herndon, VA) by centrifugation. On selected patients, isolated PBMCs were placed in culture for 72 hours or less in 10% fetal bovine serum–supplemented RPMI medium (Sigma-Aldrich) in a humidified 5% CO2 incubator at 37°C, and cells were collected at the desired times, centrifuged at 1000g (2000 rpm), washed in sterile 1 x phosphate-binding buffer (PBS), and frozen as pellets for later use. Electrophoretic mobility shift assays Nuclear extracts were prepared from fresh or cryopreserved (10% DMSO) PBMCs of ATLL patients with leukemia, total PBMCs, and magnetically isolated CD4+ cells from a healthy donor (CD4+ T Cell Isolation Kit II; Miltenyi Biotec, Bergisch-Gladbach, Germany), leukemic PBMCs from an HTLV-1–negative patient with CD4+CD25+ lymphoma, and fresh lymphoma-type ATLL tumor specimens. Human T-cell lines MT-2 (Tax-expressing, HTLV-1+) and Jurkat (ATCC) were also used as controls. Methods for nuclear protein extraction and EMSA supershift analyses in our laboratory have been described previously.34 Western blot analysis Whole cell extracts were prepared from MT-2 and Jurkat cells, frozen ATLL solid tumor specimens, and PBMCs from ATLL patients with leukemia and were frozen as pellets or were cryopreserved in 10% DMSO or fresh isolates before and after culture. Protein sample preparation and Western blot analysis were performed as previously described.34 Total protein (25-50 µg) was fractionated on 12% SDS-PAGE and transferred by electroblotting onto nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). Proteins were visualized with the enhanced chemiluminescence (ECL) developing kit (Amersham, Piscataway, NJ). Immunohistochemistry Paraffin sections (3-µm–thick) were dewaxed in xylene, rehydrated, and treated with 6% hydrogen peroxide to block endogenous peroxidase. Slides were then pressure cooked for 5 minutes in the presence of target retrieval solution (DAKO, Carpinteria, CA), blocked with avidin/biotin solution, and incubated in rabbit anti–IRF-4 antibody (M7259; DAKO) for 30 minutes in a humidity chamber. Primary antibody was removed from the slides by washing with PBS, and the slides were incubated in biotinylated antirabbit linking solution for 25 minutes, washed with PBS, and incubated in streptavidin peroxidase conjugated for 25 minutes in a humidity chamber. Finally, they were washed with PBS, stained with DAB (DAKO), washed in PBS again, submerged in 1% cupric sulfate, rinsed in H2O, and counterstained with 0.1% Fast Green. Images were visualized with an Olympus BH-2/BHTV microscope (Center Valley, PA). The H&E-stained picture (top panel, Figure 4) was visualized with a 100x/lp25 NA oil-immersion objective lens, and MUM1 (bottom panel, Figure 4) was visualized with a 50x/0.90 NA oil-immersion objective lens. Stains used were hematoxylin-eosin (H&E) and Fast Green. Imaging medium used for immunohistochemical stain was Tissue-TEK Mounting Media #6419 (Sakura Finetek, Torrance, CA). Images were taken with a Nikon Coolpix 5400 (Melville, NY) using Nikon View image acquisition software.
Antiviral therapy response in patients with ATLL and clonal persistence in long-term survivors
Twenty-eight patients included in this analysis were treated with high-dose parenteral AZT (1.5 g twice daily) and IFN-
Expression of nuclear NF-  B/Rel in primary ATLL
NF-
c-Rel expression did not correlate with tumor burden, ATLL subtype, or T-cell phenotype because 1 chronic and 2 lymphoma ATLL specimens expressed c-Rel (Table 1). In the 19 patients for whom we measured c-Rel expression, the response rate for c-Rel– patients was 66.7% (8 of 12) compared with 14.3% (1 of 7) for c-Rel+ patients, and the difference was marginally significant (P = .057; 2-sided Fisher exact test) (Table 2, left panel). Six of 7 c-Rel+ patients did not respond to AZT and IFN- . ATLL-7 was a c-Rel+ patient who achieved PR according to our criteria but who never achieved a WBC count below 18 x 109/L. All 5 patients who achieved CR were in the c-Rel– group. CR rates for c-Rel– and c-Rel+ groups were 41.7% and 0%, respectively (P = .106; 2-sided Fisher exact test).
IRF-4 expression correlates with c-Rel and antiviral therapy resistance in ATLL
c-Rel has been associated with the expression of IRF-4, which has been implicated in interferon resistance.36 IRF-4 has been shown to play an important role in T-cell function, has oncogenic activity in vitro, and is expressed in patients with aggressive lymphomas, including ATLL, of poor prognosis.37–45 IRF-4 is also thought to function as a repressor of IFN-regulated genes, and IRF-4–deficient c-Rel–/– cells are markedly sensitive to the antiproliferative effects of IFN.36,46,47 IRF-4 is readily detected in HTLV-1–infected cell lines and primary ATLL cells that express the viral oncoprotein Tax.47–49 However, uncultured primary ATLLs do not express Tax.23 Because of the observed c-Rel expression in several antiviral (IFN-
We examined IRF-4 protein expression in tumors for which sample material was available by Western blot analysis or immunohistochemistry (IHC). Data on IRF-4 expression were available for 20 of the 22 patients assessed for response to AZT and IFN-
IRF-4 was detected by Western blot in all 6 c-Rel+ tumors tested (Figure 3A–B), including a lymph node specimen (ATLL-20) analyzed by IHC (Figure 4). In all but one patient (ATLL-7), tumors were resistant to AZT and IFN- (Table 1). IRF-4 was also detected in 3 c-Rel– tumors from patients in whom antiviral therapy failed (ATLL-11, ATLL-23, ATLL-26). In contrast, no significant IRF-4 protein was detected in 9 (69%) of 13 c-Rel– tumors, including 7 from patients whose tumors were antiviral sensitive and all 5 complete responders (ATLL-1, ATLL-2, ATLL-3, ATLL-14, ATLL-21, ATLL-24, ATLL-27). In summary, almost all antiviral therapy–resistant tumors, most of which were c-Rel+, had detectable IRF-4 protein levels, whereas the antiviral-sensitive tumors (including all complete responders) were c-Rel– and lacked IRF-4 protein (Table 1). The association between IRF-4 and c-Rel expression was statistically significant (P = .03). Kaplan-Meier survival plots indicated significance and a tendency toward better survival in ATLL patients lacking c-Rel and IRF-4 in their tumors, respectively (P = .021 and P = .061, respectively; log rank test) (Figure 5).
We then analyzed whether IRF-4 was associated with Tax expression in ATLL. IRF-4 is highly expressed in Tax-positive HTLV-1–infected cells, though no significant Tax expression occurs in primary unmanipulated ATLL tumors.23,47–50 In fact, low anti-Tax antibody titers have been associated with the development of ATLL in HTLV-1 carriers.51 ATLL cells do, however, begin to express Tax spontaneously after introduction into tissue culture.23,28 We observed high IRF-4 protein levels at baseline in MT-2– and non-Tax–expressing tumors (ATLL-5, ATLL-22) (Figure 3B). However, Tax protein expression in ATLL cells (ATLL-21, ATLL-22, ATLL-24) occurred only after in vitro culture, which coincided with the expression of IRF-4. In summary, IRF-4 was expressed in the absence of Tax in primary unmanipulated antiviral-resistant ATLL cells, but it was coinduced with Tax only after culture in antiviral-sensitive tumors.
ATLL generally remains an incurable illness that recurs even after treatment with intensive chemotherapy regimens. Several studies have shown that IFN- –based regimens, in combination with either AZT or arsenic trioxide, may benefit a subset of patients with ATLL.10–13,52 The molecular mechanisms that predict response to antiviral-based therapy and the mechanisms of action of these drugs in ATLL are likely complex and involve a variety of factors. Mutations in tumor suppressors (p15, p16, p53) and overexpression of prosurvival molecules, including NF-B, survivin, and IRF-4, have been found in ATLL tumors of poor prognosis.23,24,45,53–55 Alterations of death receptor signaling (FAS, ligand, and TRAIL receptors, c-FLIP) have also been observed in ATLL cells,55–57 and IFN signaling defects have been identified in several malignancies, including T-cell lymphomas.17–19 In this study, we found that lack of response to antiviral therapy correlated with the nuclear expression of the NF-B subunit c-Rel, particularly with its putative target, IRF-4. Resistance to AZT and IFN- correlated independently with the expression of these 2 oncogenic proteins, but coexpression in the resistant tumors was common. Conversely, lack of IRF-4 and c-Rel was associated with response to antiviral therapy, particularly complete response and long-term survival. IRF-4 alone may be a more useful predictor of antiviral therapy response because it was overexpressed in resistant tumors lacking c-Rel. IRF-4 immunohistochemistry is also widely available in many centers.
NF-
We also investigated the expression of IRF-4 in primary ATLL tumors. IRF-4 has transforming and oncogenic potential in vitro39 and is overexpressed in non-Hodgkin lymphomas of poor prognosis, including aggressive-type ATLL.40–45 IRF-4 is a member of the interferon regulatory factor family of transcription factors whose expression is restricted to the lymphoid and myeloid compartments.68,69 In normal lymphocytes, IRF-4 is important in gene expression, proliferation, and differentiation.37,70 In mature human CD4+ T cells, IRF-4 is essential for cytokine production and cell survival.37 IRF-4 is also thought to function as a repressor of toll-like receptor (TLR)–dependent inflammatory cytokines and IFN-regulated genes, and IRF-4–deficient c-Rel–/– cells demonstrate unusual sensitivity to the antiproliferative effects of IFN.46,47,71 IRF-4 is constitutively upregulated in HTLV-1–infected cell lines and primary ATLL cells that express the oncoprotein Tax.47–49 However, uncultured primary ATLL does not express Tax.23 We detected IRF-4 protein in most antiviral therapy–resistant ATLL tumors tested and in one c-Rel+ partial responder, but no IRF-4 protein was detected in the tumors of the other patients responsive to AZT and IFN- IRF-4 expression in primary antiviral-resistant ATLL was independent of the HTLV-1 oncoprotein Tax, which was not detected in primary unmanipulated tumors that exhibited IRF-4. Our findings do confirm that primary ATLL cells can coexpress Tax and IRF-4—but only after they are placed in culture—and support the role of Tax in the induction of IRF-4.47,48
It is likely that NF-
Antiviral therapy proved to be effective in a subset of our leukemic patients, some of whom have been maintained in clinical remission for years. In contrast, essentially all patients with lymphoma-type ATLL have fared poorly. Similar experiences have been reported by other investigators.3 We found that the peripheral blood of long-term ATLL survivors in stable remission continued to have low levels of detectable T-cell clones. Other investigators have demonstrated that persistence of tumor clones in ATLL patients invariably correlated with disease relapse.73 These data indicate that AZT and IFN-
Contribution: J.C.R. performed most of the work, analyzed the data, and coauthored the paper. P.R. performed clonality assays. L.R. provided resources and reagents. I.R. performed statistical analysis. C.B. contributed specimens. C.P. contributed and processed specimens. G.B. performed immunohistochemical analysis. L.T. performed experiments. V.A. assisted in NF- B experiments. E.H. contributed reagents. I.L. assisted in gene expression and data analysis. W.J.H. designed the research, analyzed the data, and coauthored the paper. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: William J. Harrington Jr, University of Miami Miller School of Medicine, Sylvester Comprehensive Cancer Center, Rm 3400 (D8-4), 1475 NW 12th Ave, Miami, FL 33136; e-mail: wharring{at}med.miami.edu.
Supported by National Institutes of Health grants UO1-CA-070058 (AIDS Malignancy Consortium), CA-082274, and CA-10521.
Submitted July 20, 2006; accepted November 11, 2006.
Prepublished online as Blood First Edition Paper, November 30, 2006
DOI: 10.1182/blood-2006-07-036368
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
