|Home | About | Journals | Submit | Contact Us | Français|
Peripheral T-cell lymphomas (PTCLs) are fatal in the majority of patients and novel treatments, such as protein tyrosine kinase (PTK) inhibition, are needed. The recent finding of SYK/ITK translocations in rare PTCLs led us to examine the expression of Syk PTK in 141 PTCLs. Syk was positive by immunohistochemistry (IHC) in 133 PTCLs (94%), whereas normal T cells were negative. Western blot on frozen tissue (n=6) and flow cytometry on cell suspensions (n=4) correlated with IHC results in paraffin. Additionally, western blot demonstrated that Syk-positive PTCLs show tyrosine (525/526) phosphorylation, known to be required for Syk activation. Fluorescence in situ hybridization showed no SYK/ITK translocation in 86 cases. Overexpression of Syk, phosphorylation of its Y525/526 residues and the availability of orally available Syk inhibitors suggest that Syk merits further evaluation as a candidate target for pharmacologic PTK inhibition in patients with PTCL.
Peripheral T-cell lymphomas (PTCLs) remain a major treatment problem among all lymphomas because of their high mortality rate and the minimal effectiveness of conventional chemotherapy.1 Novel therapeutic strategies, such as inhibiting protein tyrosine kinases (PTKs), might improve the outlook toward the treatment of patients with PTCL. Recently, a t(5;9)(q33;q22) translocation2 was reported in a subgroup of PTCL with follicular involvement,3 resulting in overexpression of the SYK gene under the control of the ITK promoter. SYK encodes a cytoplasmic PTK, which is important in proliferation and prosurvival signaling4–7 and is expressed in a variety of hematopoietic cells, including normal B lymphocytes8 and most B-cell lymphomas.5,9–12 Normal peripheral T cells, however, generally lack Syk protein expression.13
In the current work, we demonstrate that Syk is overexpressed in the majority of PTCLs, despite the absence of SYK/ITK translocations in most cases. As one orally available Syk inhibitor14 is already in clinical trial for B-cell lymphomas, Syk merits further evaluation as a possible therapeutic target in patients with PTCL as well.
We studied specimens from 141 patients with PTCL diagnosed by WHO criteria.15 There were 86 men and 55 women of a mean age of 59 years (range, 5–88 years). The study was approved by the Institutional Review Board and the Biospecimens Committee of Mayo Clinic. All patients provided informed consent for the use of their tissues for research purposes.
Paraffin tissue microarrays were constructed as described previously.16 In cases with insufficient tissue, whole-tissue sections were analyzed. Slides were pretreated in 1 mM EDTA buffer at pH 8.0 for 30 min at 98 °C (PT Module; Lab Vision, Fremont, CA, USA) and then stained for Syk with a rabbit polyclonal antibody (C-20, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Dual Link Envision+/DAB+ (Dako, Carpinteria, CA, USA) was used for detection. Tumors were considered positive for Syk when >30% of the neoplastic cells demonstrated Syk staining. Slides were visualized through an Olympus BX51 microscope (Olympus, Melville, NY, USA) and photographed with an Olympus DP71 camera using Olympus DP manager image acquisition software.
Protein lysates prepared from frozen tissue sections of six PTCLs and from the B-cell lymphoma cell line, Raji, were separated by polyacrylamide gel (Bio-Rad, Hercules, CA, USA) electrophoresis, transferred to PVDF membranes (Bio-Rad) and incubated for 1 h with primary antibodies as follows: Syk (1:500; N-19, Santa Cruz), phospho-Syk (Tyr525/526, 1:1000; no. 2711, Cell Signaling Technology, Danvers, MA, USA) and actin (1:1000; C-11, Santa Cruz).
Flow cytometric immunophenotyping was performed on thawed, washed cells as described previously.17 Briefly, cells were stained with fluorochrome-conjugated antibodies (Becton Dickinson/Pharmingen, San Jose, CA, USA) to CD3 (peridinin chlorophyll protein), CD5 (phycoerythrin) and CD19 (PE-Cy7). Stained cells were washed, fixed and permeabilized (Caltag Fix and Perm; Caltag/Invitrogen, Eugene, OR, USA) and then stained with anti-Syk (fluorescein isothiocyanate). Cells were analyzed on a FACSCanto instrument (Becton Dickinson) and data were analyzed using FACSDiva Software (Becton Dickinson).
Interphase fluorescence in situ hybridization (FISH) was performed on tissue microarray or whole-tissue sections as described previously,16 using dual fusion (D-FISH) SpectrumOrange- and SpectrumGreen-labeled DNA probes that hybridize to regions spanning the SYK and ITK breakpoints involved in the t(5;9)(q33;q22) translocation. A minimum of 50 cells were scored per case. Control material carrying the translocation was kindly provided by Dr B Streubel (Vienna, Austria).
We evaluated Syk expression in reactive and neoplastic T cells by immunohistochemistry (IHC) using a polyclonal antibody against the C terminus of Syk. Although T cells in reactive tonsil, lymph node and spleen were negative (Figure 1a), IHC demonstrated cytoplasmic Syk expression in 133/141 (93%) PTCLs studied. These included 35/35 (100%) AITLs (angioimmunoblastic T-cell lymphomas; Figure 1b), 62/66 (94%) PTCL-Us (PTCLs, unspecified; Figure 1c), 6/6 (100%) anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphomas (ALCLs), 11/12 (92%) systemic ALK-negative ALCLs (Figures 1d and e), 3/3 (100%) cutaneous ALCLs, 4/4 (100%) mycosis fungoides (nodal involvement), 1/2 (50%) enteropathy-associated T-cell lymphoma, 4/5 (80%) extranodal NK/T-cell lymphomas, nasal type (NKTLs) 4/5 (80%) hepatosplenic T-cell lymphomas (Figure 1f), 2/2 (100%) subcutaneous panniculitis-like T-cell lymphomas and 1/1 (100%) T-prolymphocytic leukemia. All eight Syk-negative cases were extranodal, including ALK-negative ALCLs (Figure 1d), enteropathy-associated T-cell lymphomas, hepatosplenic T-cell lymphomas (Figure 1e), NKTLs and PTCL-Us (four cases). Seven of these had a cytotoxic phenotype by IHC.
Because Syk expression was found in a greater proportion of PTCLs than previously reported,10 we corroborated the IHC results using western blotting. Reactive splenic lymphocytes were sorted by flow cytometry into B-cell, αβ T-cell and γδ T-cell populations. B-cell lysates demonstrated a 72 kDa band corresponding to Syk, whereas T-cell lysates were negative (Figure 2a). Analysis of frozen tumor tissue lysates (Figure 2b) showed cases that were Syk-negative by IHC to be negative by western blot (PTCL-Us, two cases) as well. All four cases that were Syk-positive by IHC were positive by western blot (two AITLs, one ALK-negative ALCL and one PTCL-U). To evaluate the activation status of Syk in PTCLs, we probed western blots with phospho-specific anti-Syk (Tyr525/526); these tyrosine residues reside in the catalytic domain of Syk kinase and their phosphorylation is necessary for Syk activity.18 Syk was phosphorylated at these residues in 4/4 Syk-positive PTCLs tested (Figure 2b).
Because the lysates used for western blot might contain Syk derived from non-tumor cells as well as PTCLs, we also evaluated Syk expression by flow cytometry. Reactive T cells from peripheral blood, lymph node and spleen were negative for Syk, whereas reactive B cells were positive (not shown). By using appropriate gating strategies in PTCLs with an aberrant T-cell phenotype, we could assess Syk expression specifically in the neoplastic T cells in four cases. The tumor cells demonstrated Syk expression in three cases (Figure 2c; see also Figure 1c). One case of hepatosplenic T-cell lymphoma was Syk-negative by flow cytometry (Figure 2d) as well as IHC (Figure 1f).
To determine the relationship between Syk overexpression in our series and the t(5;9)(q33;q22) SYK/ITK translocation, we evaluated cases using D-FISH probes for SYK and ITK. Despite appropriate fusion signals in control tissue with the translocation (not shown), no evidence of t(5;9)(q33;q22) was identified in 86 informative study cases of PTCL (84 of which were positive for Syk by IHC). These 86 cases included 23 AITLs, 38 PTCL-Us, 13 ALCLs and 12 other cases, a distribution similar to that in the overall study set. Additional copies of SYK (3–6 signals) were identified in only four cases, including two ALK-negative ALCLs (both Syk protein-positive by IHC) and two PTCL-Us (one Syk-positive and one Syk-negative). The FISH probes used did not allow distinction between gene amplification and polysomy as the cause for additional SYK signals. None of the cases studied had the characteristic features of PTCLs with follicular involvement described by de Leval et al.,3 which were seen in 3/5 previously reported cases with SYK/ITK translocation.2 Based on our findings, translocations or additional copies of SYK do not appear to be the mechanisms leading to Syk protein overexpression in most Syk-positive PTCLs.
Syk has been suggested as a potential therapeutic target for PTCL by Mahadevan et al.,19 but previous data on Syk expression in T-cell lymphomas are limited and somewhat conflicting. A small study found Syk in only 2/19 PTCLs by IHC, including 1/8 PTCL-U and 1/1 mycosis fungoid (weak staining).10 The higher positivity rate found by us might be due to differences in the antibodies used, or due to unknown differences in the patient populations studied. Syk expression and Syk kinase activity have been reported to be decreased in lysates of cutaneous T-cell lymphoma cells isolated from peripheral blood (n=4),18 a source not evaluated in our study. This difference in site might account for our finding that Syk was expressed in 4/4 cases of lymph node involvement by mycosis fungoides. Other previous studies have shown Syk overexpression in SYK-translocated cases,2 Syk upregulation in adult T-cell leukemia/lymphoma cell lines20 and a relative increase in SYK expression in ALK-positive ALCLs.21
Several comments regarding the interpretation of our findings are warranted. First, IHC of reactive lymphoid tissue showed Syk-positive cells to outnumber CD20-positive cells in the paracortex (Figure 1a). Most lymphocytes appeared negative for Syk. By morphology and distribution, many of the positive cells appeared to be histiocytes and/or dendritic cells, which are among the hematopoietic cell types that express Syk.22 Without double immunostaining, the presence of a minimal population of normal Syk-positive T cells cannot be entirely excluded. However, such a population was not identified by flow cytometry, which is a highly sensitive method of detection. Second, unlike most normal T cells, NK cells have been reported to express Syk.23 Although we did not include tumors of known NK-cell origin in our series, we did include cases of NKTLs, which may be of either NK- or T-cell origin.15 Four out of five NKTLs were Syk-positive by IHC. If these four positive cases were of NK-cell origin, the observed Syk positivity might reflect constitutive expression in this cell type rather than lymphoma-associated overexpression. Finally, as mentioned above, tumor lysates subjected to western blot would be expected to contain some protein from admixed non-neoplastic cells. The phosphorylation status of Syk in the admixed B cells present in PTCLs such as AITLs is unknown. In B-cell lymphomas such as follicular lymphoma, tumor-infiltrating non-neoplastic B cells appear to demonstrate lesser Syk phosphorylation than the tumor cells on stimulation.24 However, as phosphorylation of Syk is a physiologic event in B-cell receptor-mediated signaling,8 we cannot exclude the possibility that some of the phospho-Syk detected by western blot of PTCL samples (Figure 2b) was derived from admixed B cells.
Patients with PTCL are usually treated with CHOP or more intensive regimens, generally with minimal effectiveness, and new therapeutic strategies are needed.25 In this study, we demonstrate that Syk PTK is overexpressed in the majority of PTCLs. A phase II clinical trial of an orally available Syk inhibitor is underway for B-cell lymphomas. Overexpression of Syk, phosphorylation of its Y525/526 residues and the availability of pharmacologic inhibitors suggest that Syk may be a suitable target for PTK inhibition in PTCL patients. Studies of the effect of Syk inhibitors on T-cell lymphoma cell lines are warranted to evaluate this possibility further.
We acknowledge the support from the Iowa/Mayo Lymphoma SPORE grant from the National Cancer Institute (P50 CA97274). In addition, we thank Ms Connie Lesnick for help with flow cytometry, Dr B Streubel for providing control specimens with the SYK/ITK translocation and Ms Monica Kramer and Ms Carrie Stevenson for administrative assistance.