Clinical Chemistry / Serum TK1 and Prognosis in CLL. High Serum ... Key Words: Chronic lymphocytic leukemia; Serum thymidine kinase 1; Prognostic factors.
Clinical Chemistry / Serum TK1 and Prognosis in CLL
High Serum Thymidine Kinase 1 Level Predicts Poorer Survival in Patients With Chronic Lymphocytic Leukemia Sergej N. Konoplev, MD, PhD,1 Herbert A. Fritsche, PhD,1 Susan O’Brien, MD,2 William G. Wierda, MD,2 Michael J. Keating, MD,2 Terrie G. Gornet,1 Susan St Romain, MT(ASCP),1 Xuemei Wang, MS,3 Kedar Inamdar, MD,1 Malisha R. Johnson, DO,1 L. Jeffrey Medeiros, MD,1 and Carlos E. Bueso-Ramos, MD, PhD1 Key Words: Chronic lymphocytic leukemia; Serum thymidine kinase 1; Prognostic factors DOI: 10.1309/AJCPHMYT93HUIZKW
Abstract Serum thymidine kinase 1 (TK1) levels have been reported to have prognostic significance in patients with chronic lymphocytic leukemia (CLL). Until recently, serum TK1 levels were assessed using inconvenient radioenzyme assays. In this study, we used a novel chemiluminescence assay to assess serum TK1 levels in patients with CLL at the time of first examination. We show that high serum TK1 levels predict poorer overall survival and correlate with unmutated immunoglobulin variable region genes, CD38 and ZAP-70 expression, and subsequent risk of developing large B-cell lymphoma (Richter syndrome). Similar findings were observed in a subset of patients treated with current fludarabine-based chemotherapy regimens. We suggest that serum TK1 levels analyzed using this convenient chemiluminescence assay may be useful in the risk assessment of patients with CLL.
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Am J Clin Pathol 2010;134:472-477 DOI: 10.1309/AJCPHMYT93HUIZKW
Chronic lymphocytic leukemia (CLL) is the most common hematologic neoplasm of adults in Western countries. In the United States, more than 15,000 new cases are diagnosed annually, and more than 4,000 patients die annually of this disease.1 The clinical course of patients with CLL is variable; most patients have a long survival, but in some patients, early disease progression develops.2 This clinical heterogeneity justifies the need for reliable prognostic markers that allow therapy to be tailored to a particular patient. In many neoplasms, the clinical course is related to the proliferative activity of the neoplastic cells. Therefore, indicators of proliferation are attractive candidates as prognostic markers. Traditionally, the proliferative component of CLL has been underappreciated, with more focus on apoptosis and other pathogenic mechanisms. However, more recently, the importance of proliferation in CLL pathogenesis has been recognized. One indicator of cell proliferation in CLL is thymidine kinase (TK), a cellular enzyme involved in a salvage pathway for DNA synthesis. There are 2 isoforms of this enzyme: TK1 and TK2. TK1 is found in the cytoplasm of dividing cells and is absent in resting cells. TK2 is located in the mitochondria of resting cells.3,4 In 1984, Kallander and colleagues5 assessed serum TK1 levels in patients with CLL using a radioenzymatic assay (REA) and demonstrated that higher serum TK1 levels correlated with progressive disease and advanced Rai stage. More recently, Hallek and colleagues,6 using similar methods, demonstrated that the serum TK1 levels predicted progression-free survival in patients with CLL independently from other known prognostic factors, such as WBC count and presence of lymphadenopathy. Hallek and others7 confirmed that the serum TK1 level was helpful © American Society for Clinical Pathology
Clinical Chemistry / Original Article
in identifying a subgroup of patients with CLL at high risk for disease progression. Magnac et al,8 using REA methods, identified a strong correlation between high serum TK1 levels and unmutated immunoglobulin variable heavy chain (IGHV) genes. The last observation raised a question as to whether the serum TK1 level is an independent prognostic marker or simply reflects mutational status of the IGHV gene. In addition, as previous studies analyzed data for patients with CLL not treated with current therapeutic modalities, it is not clear whether the previous observations remain valid in the current era of chemoimmunotherapy for patients with CLL. In this study, we used a chemiluminescence immunoassay (CLIA) method to assess serum TK1 levels in untreated patients with CLL. Serum TK1 levels were also correlated with other clinical and laboratory parameters.
Materials and Methods Patient Study Group After receiving approval from the institutional review board, we searched the files of our institution from January 2000 through June 2008 for cases of CLL, previously untreated, with serum samples available for TK1 analysis. The original diagnoses were confirmed by review of bone marrow aspirate smears and biopsy specimens, the results of flow cytometry immunophenotypic analysis, and immunohistochemical studies performed on bone marrow biopsy and/or clot sections. Detection of Serum TK1 TK1 was analyzed in baseline serum samples of all patients before therapy. During the initial stage of this project, we analyzed serum samples for TK1 in the first 50 patients in parallel using a standard REA and a novel CLIA. REA is based on the conversion of iodine 125 (125I)-labeled deoxyuridine to 125I deoxyuridine monophosphate and was performed on the Prolifigen TK REA, DiaSorin, Stillwater, MN, as described previously.9 Briefly, 500 μL of radioactive substrate was added to 20 μL of patient serum sample and incubated for 4 hours at 37°C in a water bath. A TK-REA separator tablet was added to each sample tube and incubated at room temperature for 1 hour. Tube contents were washed for a total of 4 washes and then counted on a gamma counter. A series of standards and controls were set up simultaneously with patient samples to allow the construction of a standard curve and monitor for interbatch and intrabatch variation. The CLIA, which is easy to perform, is based on TK1 converting AZT (3′-azido–3′deoxythymidine) to AZTMP (3′-azido–3′-deoxythymidine monophosphate) and is
commercially available (DiaSorin). The correlation between these methods was excellent (R = 0.9764; y = 1.0158x + 3.2691). For this reason, for the remainder of this study, we used only the CLIA method to detect serum TK1 levels. There was no significant difference between clinical and laboratory parameters for the first and second groups of patients, which allowed us to combine data for both groups of patients in the analysis. CD38 Expression CD38 expression was evaluated in all patients by flow cytometric immunophenotypic analysis of bone marrow aspirate specimens at the time of initial diagnosis. The percentage of CD38+ B cells was determined by the fraction of CD19+ cells that coexpressed CD38. A 30% cutoff was used for positive CD38 expression. ZAP-70 Expression by Immunohistochemical Analysis ZAP-70 expression was evaluated by immunohistochemical analysis using paraffin-embedded bone marrow tissue sections and a monoclonal murine anti–ZAP-70 antibody (Upstate Cell Signaling Solutions, Charlottesville, VA), as previously described.10 A positive result was determined by observing ZAP-70 expression by B cells. ZAP-70 is expressed strongly by T cells alone and served as an internal control. Somatic Mutation Status of the IGHV Gene Sequence analysis of the IGHV genes was performed using total RNA extracted from bone marrow aspirates or peripheral blood specimens as described previously. To determine the level of somatic mutation, sequences were aligned with the germline sequences in the V-BASE 2 database.11 The IGHV mutation status was designated as unmutated if there were fewer than 2% mutations (≥98% homology to germline sequences) or as mutated if there were 2% or more mutations (
