Thymidine kinase 1 - Nature

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Jan 25, 2007 - 7Department of Hematology and Oncology, St. Anna. Children's Hospital, Vienna, Austria;. 8Department of Pediatric Hematology and Oncology ...

Letters to the Editor

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Pediatric Hematology–Oncology, IRCCS Policlinico San Matteo, Pavia, Italy; 6 Department for Pediatric Hematology/Oncology, Kinderklinik Medizinische Hochschule Hannover, Hannover, Germany; 7 Department of Hematology and Oncology, St. Anna Children’s Hospital, Vienna, Austria; 8 Department of Pediatric Hematology and Oncology, University Hospital, Motol, Prague, Czech Republic; 9 Department of Pediatric Hematology, Oncology and Hemostasis, Johann Wolfgang Goethe University Hospital, Frankfurt am Main, Germany; 10 Department of Paediatric Haematology/Oncology, University Children’s Hospital, Eberhard-Karls-University, Tuebingen, Germany; 11 Department of Pediatric Hematology and Oncology, Dr von Haunersches Kinderspital, Children Hospital of the Ludwig-Maximilians-University of Munich, Munich, Germany; 12 Department of Pediatrics, University of Kiel, Kiel, Germany; 13 Department of Pediatric Hematology and Oncology, University Hospital, Giessen, Germany; 14 Department of Pediatrics, University of Bologna, S. Orsola Hospital, Bologna, Italy; 15 Section of Pediatrics, Department of Women’s and Children’s Health, Uppsala University, Uppsala, Sweden; 16 Department of Pediatric Hematology, Our Lady’s Hospital for Sick Children, Dublin, Ireland; 17 Dutch Childhood Oncology Group, The Hague, Erasmus University Medical Center-Sophia Children’s Hospital, Rotterdam, the Netherlands and 18 Department of Pediatrics, Skejby Hospital, Aarhus University, Aarhus, Denmark E-mail: [email protected]

References 1 Locatelli F, Nollke P, Zecca M, Korthof E, Lanino E, Peters C et al. Hematopoietic stem cell transplantation (HSCT) in children with juvenile myelomonocytic leukemia (JMML): results of the EWOGMDS/EBMT trial. Blood 2005; 105: 410–419. 2 Yoshimi A, Bader P, Matthes-Martin S, Stary J, Sedlacek P, Duffner U et al. Donor leukocyte infusion after hematopoietic stem cell transplantation in patients with juvenile myelomonocytic leukemia. Leukemia 2005; 19: 971–977. 3 Bosi A, Laszlo D, Labopin M, Reffeirs J, Michallet M, Gluckman E et al. Second allogeneic bone marrow transplantation in acute leukemia: results of a survey by the European Cooperative Group for Blood and Marrow Transplantation. J Clin Oncol 2001; 19: 3675–3684. 4 Manabe A, Okamura J, Yumura-Yagi K, Akiyama Y, Sako M, Uchiyama H et al. Allogeneic hematopoietic stem cell transplantation for 27 children with juvenile myelomonocytic leukemia diagnosed based on the criteria of the International JMML working group. Leukemia 2002; 16: 645–649. 5 Yusuf U, Frangoul HA, Gooley TA, Woolfrey AE, Carpenter PA, Andrews RG et al. Allogeneic bone marrow transplantation in children with myelodysplastic syndrome or juvenile myelomonocytic leukemia: the Seattle experience. Bone Marrow Transpl 2004; 33: 805–814. 6 Smith FO, King R, Nelson G, Wagner JE, Robertson KA, Sanders JE et al. Unrelated donor bone marrow transplantation for children with juvenile myelomonocytic leukaemia. Br J Haematol 2002; 116: 716–724. 7 Locatelli F, Niemeyer C, Angelucci E, Bender-Go¨tze C, Burdach S, Ebell W et al. Allogenic bone marrow transplantation for chronic myelomonocytic leukemia in childhood: a report from the European working group on myelodysplastic syndrome in childhood. J Clin Oncol 1997; 15: 566–573. 8 Chang YH, Jou ST, Lin DT, Lu MY, Lin KH. Second allogeneic hematopoietic stem cell transplantation for juvenile myelomonocytic leukemia: case report and literature review. J Pediatr Hematol Oncol 2004; 26: 190–193.

Thymidine kinase 1 – A prognostic and diagnostic indicator in ALL and AML patients

Leukemia (2007) 21, 560–563. doi:10.1038/sj.leu.2404536; published online 25 January 2007

Acute leukemias are a collection of diseases represented by a predominance of undifferentiated or immature lymphoid (acute lymphoid leukemia (ALL)) or myeloid (acute myelogenous leukemia (AML)) precursors in bone marrow and peripheral blood. Malignant cells replace normal marrow cells and disrupt routine blood cell function, leading to death within weeks to months if left untreated. Chemotherapeutic advances have dramatically altered the management of patients with acute leukemias. Both ALL and AML patients can reasonably expect an extended disease-free survival period. Notwithstanding the improvements made in the treatment of these diseases, some patients remain at risk of relapse.1 The ability to monitor patient response to treatment would enhance disease management. The putative cancer marker, thymidine kinase (TK), has been shown to be elevated in the serum of patients with a variety of human malignancies.2 TK is a pyrimidine salvage pathway enzyme involved in DNA synthesis and repair. In the presence of adenosine triphosphate, TK catalyzes the conversion of thymidine to thymidine monophoLeukemia

sphate (dTMP), dTMP is subsequently phosphorylated to its triphosphate analog (dTTP) by TK.3 TK is present in human cells in two major forms, TK1 and TK2. During the G1/S transition of normal cells, TK1 levels increase by 10- to 20-fold. TK1 levels remain elevated in the cell until M phase, at which time TK1 is rapidly degraded.4 The rate of degradation appears to change in a cell-cycle-dependent manner, resulting in the increased observed levels of TK1 activity. Cancer cells are known to have lost cell-cycle control of TK1, which leads to increased levels of TK1 in these cells and could possibly explain the elevations found in serum. TK2, the other major TK isozyme, is of mitochondrial origin and its levels are independent of the cell cycle and remain constant in both cancer cells and normal cells, as well as sera.5 It has been proposed that serum TK1 levels (i) may indicate patients’ response to therapy, (ii) may serve as a prognostic indicator and (iii) may reflect the aggressiveness of leukemic cells.6 The mechanism of how TK1 enters the blood stream is not yet fully understood; nevertheless, experimental evidence suggests that screening serum for elevated TK1 levels may prove to be a simple and effective means of detecting and monitoring malignant disease. The level of serum TK1 in patients with several types of cancer has been shown to correlate with the stage of the disease and repeated measurements also reflect relapses and remissions.7

Letters to the Editor

561 TK1 has also been shown to be of prognostic value in many solid tumors, including breast cancer, prostate cancer, bladder carcinoma and small-cell carcinoma of the lung.3,6 Serum TK1 levels correlate strongly with cancer stage and its aggressiveness. In this communication, we report that measuring the serum TK1 levels of ALL and AML patients would provide a method for monitoring patient response to treatment in acute leukemic diseases and potentially other non-solid tumors. This study consists of several phases. In brief, we first compared the serum TK1 levels of ALL patients with non-cancer patients. Second, sera from the ALL patients were grouped according to clinical status (pre-treatment, relapse, remission) and analyzed against non-cancer controls. Finally, TK1 levels in sera from individual patients both ALL and AML were sequentially followed for over a year. In the initial study, TK1 levels were measured in serum samples from ALL patients (19 females/14 males) and healthy individuals (35 males/14 females; Figure 1). Sera samples from both sample groups were measured using a radioassay as well as an immunoassay employing a TK1-specific monoclonal antibody (mAb) developed in our laboratory.8 Following a natural logarithm transformation to normalize the distribution of these data, the Student’s t-test and one-way ANOVA were performed. The increase in the mean serum TK1 levels between the non-cancer control individuals and the ALL patients was statistically significant (Po0.05). Covariance analysis was performed and it was determined that gender did not significantly affect the serum TK1 levels (P40.05). TK1 activity was not significantly different in age, blood type, white blood cell count and race of the ALL patients, as determined by

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Figure 1 Serum TK1 activity in ALL and non-cancer patients. (a) TK1 activity was measured by radioassay. (b) TK1 levels were detected by immunoassay. (a and b) Data are averages from four separate experiments (Po0.01).

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one-way covariance analysis or Student’s t-test (P40.05) (data not shown). Comparison of the radioassay (data transformed to natural logarithm to normalize the distribution of the data) and the immunoassay absorbance data using the Pearson correlation indicates a strong correlation between TK1 measurements for the assays. The Pearson correlation showed that the correlation was 0.806 (Po0.05), demonstrating that the margin of error is an acceptable value between the radioassay and the immunoassay. Following the encouraging results of the initial phase, these same 33 patients were analyzed according to the stage of their disease. Figure 2 shows the mean serum TK1 activity that was determined for the ALL patients who had not yet started treatment, patients who were experiencing relapse and patients who were in remission. Relapse patients had a mean serum TK1 activity 82 times higher than the non-cancer control serum TK1 level. These differences were statistically significant (Po0.05). A significant difference (Po0.05) also existed between the means of the serum TK1 activity of the pre-treatment ALL patients and between the patients who had relapsed after treatment. However, no difference (P40.05) existed between the control group and the ALL patients currently in remission. These results indicate the prognostic value of measuring serum TK1 levels. Having obtained the above results, we decided to monitor intra-patient TK1 levels. Serial serum samples obtained from nine patients (four ALL and five AML) were collected over a timeline up to 360 days, and presented as a percentage of the maximum serum TK1 activity. Serum activity was elevated in every patient at the onset of treatment for ALL. As treatment progressed, serum TK1 activity dropped and remained low if the treatments were successful and the patients went into remission. This pattern can be seen in ALL patients A, B and C (Figure 3a– c). The pattern for patient D (Figure 3d) showed an initial drop in TK1 activity as treatment began, but increased as patient D relapsed into progressive disease. Samples from AML patients (Figure 4) A, B and C were obtained as they began treatment. AML patients D and E had already begun treatment when the initial samples were collected. Results are shown in Figure 4. These patients followed the same trend seen with the ALL patients that were analyzed. The serum TK1 activity of AML patients C, D and E initially decreased (Figure 4c–e). However, all three of these patients relapsed into progressive disease, which corresponded to a substantial increase in serum TK1 activity in each patient.

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Figure 2 Serum TK1 activity in pre-treatment, relapsed, remission and control patients. TK1 activity was measured by radioassay. Data are averages from four separate experiments. Leukemia

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Figure 3 Percentage of TK1 activity of maximum in ALL patients’ samples. (a) patient A, (b) patient B, (c) patient C, (d) patient D. Data are averages from four separate experiments. All patients were well at the conclusion of each respective study period.

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Figure 4 Percentage of TK1 activity of maximum in AML patients’ samples. (a) patient A, (b) patient B, (c) patient C, (d) patient D, (e) patient E. Arrows indicate patient death. If no arrow is present, patient survived beyond the respective study period. Data are averages from four separate experiments.

The current treatments used for acute leukemia continue to increase the chance of survival for the patient. However, there are few tools available for clinicians to monitor the progress of the patient during and following treatment. These results indicate serum TK1 activity in ALL and AML is an accurate indicator of response to treatment and stage of disease. The serum TK1 activity dropped significantly in Leukemia

each patient who successfully responded to treatment and went into remission. In each patient that relapsed into progressive disease, the serum TK1 activity began to increase. These data suggest that by monitoring serum TK1 activity, the clinician would be able to determine whether or not the patient is responding positively to treatment. Long-term monitoring of the serum TK1 activity would also allow the

Letters to the Editor

563 clinician to detect possible relapse in patients if TK1 levels start to increase. A minor limitation of measuring serum TK1 activity is the substantial variance between patients. This variation between individuals prevents direct comparison between patients. For this reason, we presented the changes in serum TK1 activity for the ALL and AML patients as a percentage of the maximum measured for each individual (Figures 3 and 4). Our results show that the changes in serum TK1 activity are only useful for prognosis when compared with the individual’s previous samples. Therefore, a baseline level of serum TK1 activity must be established for each individual if serum TK1 is to be used as a prognostic tool in leukemia, similar to the method currently used in monitoring prostate-specific antigen in men. Variations in TK1 levels between serial samples from the same individual are directly reflective of the response to treatment, indicating that TK1 measurements can accurately predict patient prognosis. Our results comparing the mean serum TK1 levels of ALL patients with healthy individuals indicated statistically significant differences between the two groups. This confirms the reality of measuring serum TK1 levels as a diagnostic tool. Statistical significance between serum TK1 levels in ALL patients of different stages confirms TK1 as a possible prognostic tool. This study also indicates that the immunoassay, utilizing the TK1-specific mAb, is an accurate method of measuring serum TK1 levels. The development of an immunoassay to measure serum TK1 levels would be more practical and efficient for use in a clinical setting. Through continued investigation of TK1, we hope to provide clinicians with a valuable tool for earlier detection and improved evaluation of treatment success.

Acknowledgements We thank KEM Baillie and JM Bridges for providing the serial blood samples from the ALL and AML patients. We also thank the Biological Carcinogenesis Branch for providing blood samples.

KL O’Neill, F Zhang, H Li, DG Fuja and BK Murray Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, USA E-mail: [email protected] References 1 Kantarjian HM. Adult acute lymphocytic leukemia: critical review of current knowledge. Am J Med 1994; 97: 176–184. 2 O’Neill KL, Buckwalter MR, Murray BK. Thymidine kinase: diagnostic and prognostic potential. Expert Rev Mol Diagn 2001; 1: 428–433. 3 Hannigan BM, Barnett YA, Armstrong DB, McKelvey-Martin VJ, McKenna PG. Thymidine kinases: the enzymes and their clinical usefulness. Cancer Biother 1993; 8: 189–197. 4 Sherley JL, Kelly TJ. Regulation of human thymidine kinase during the cell cycle. J Biol Chem 1988; 263: 8350–8358. 5 Johansson M, Karlsson A. Cloning of the cDNA and chromosome localization of the gene for human thymidine kinase 2. J Biol Chem 1997; 272: 8454–8458. 6 Zhang J, Jia Q, Zou S, Zhang P, Zhang X, Skog S et al. Thymidine kinase 1: a proliferation marker for determining prognosis and monitoring the surgical outcome of primary bladder carcinoma patients. Oncol Rep 2006; 15: 455–461. 7 He Q, Zhang P, Zou L, Li H, Wang X, Zhou S et al. Concentration of thymidine kinase 1 in serum (S-TK1) is a more sensitive proliferation marker in human solid tumors than its activity. Oncol Rep 2005; 14: 1013–1019. 8 Zhang F, Shao X, Li H, Robison JG, Murray BK, O’Neill KL. A monoclonal antibody specific for human thymidine kinase 1. Hybridoma 2001; 20: 25–34.

Phosphoproteomic analysis identifies the M0-91 cell line as a cellular model for the study of TEL-TRKC fusion-associated leukemia

Leukemia (2007) 21, 563–566. doi:10.1038/sj.leu.2404555; published online 25 January 2007

The TEL-TRKC gene fusion associated with the t(12;15)(p13;q25) translocation has been implicated in both hematological (acute myeloid leukemia (AML))1 and non-hematological malignancies (congenital fibrosarcoma, congenital mesoblastic nephroma and secretory breast carcinoma). In AML, the TEL-TRKC (TEL-TRKC(L)) derives from the in-frame fusion of exons 1–4 of TEL to exons 13–18 of TRKC. In contrast, the TEL-TRKC variant associated with solid tumors (TEL-TRKC(F)) contains exons 1–5 of the TEL gene. Activation of both the RAS-MAPK and PI3K-AKT pathways by TEL-TRKC contributes to oncogenic signaling in transfected NIH3T3 cells.2 However, no human cell lines are available to study the TEL-TRKC fusion. In this study, we screened over 40 AML cell lines for constitutive phosphorylation of STAT5 by Western blot. The M0-91, an AML-M0-derived cell line3, showed constitutive tyrosine phosphorylation of STAT5 (Figure 1a). To identify protein tyrosine kinases responsible for the constitutive phos-

phorylation of STAT5 in M0-91, cell lysates were trypsindigested, and phosphopeptides were immunoprecipitated with phosphotyrosine antibody (pY-100), and analyzed by LC-MS/MS mass spectrometry.4,5 LC-MS/MS mass spectrometry identified 393 phosphotyrosine sites in 265 proteins (Supplementary Table 1). Among these proteins, over 15 tyrosine kinases were tyrosine-phosphorylated (Figure 1b). Multiple tyrosinephosphorylated peptides corresponded to either TRKB or TRKC, including three tyrosines in the activation loop. Thus, either TRKB or TRKC could be aberrantly activated in M0-91 cells. While full-length TRKB/C have a molecular weight of 140–145 kDa, we observed a 50 kDa by Western blot with a pan-TRK antibody in M0-91 cells (Figure 1c). In addition, we observed tyrosine-phosphorylated peptides deriving from the TEL protein (Supplementary Table 1). TEL is a member of the ETS family transcription factor and is essential for hematopoiesis. It is a frequent target of chromosomal translocations in human cancers, and one of its fusion partners is TRKC.1 To determine whether a chimeric TEL-TRKC transcript was present, we performed 30 rapid amplification of complementary DNA (cDNA) ends on the sequence encoding the HLH domain of TEL. Sequence analysis of the resultant product revealed that the Leukemia

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