Letters to the Editor 560 5 Pediatric Hematology–Oncology, IRCCS Policlinico San References Matteo, Pavia, Italy; 6 Department for Pediatric Hematology/Oncology, 1 Locatelli F, Nollke P, Zecca M, Korthof E, Lanino E, Peters C et al. Kinderklinik Medizinische Hochschule Hannover, Hannover, Hematopoietic stem cell transplantation (HSCT) in children with Germany; 7 juvenile myelomonocytic leukemia (JMML): results of the EWOG- Department of Hematology and Oncology, St. Anna MDS/EBMT trial. Blood 2005; 105: 410–419. Children’s Hospital, Vienna, Austria; 2 Yoshimi A, Bader P, Matthes-Martin S, Stary J, Sedlacek P, Duffner 8 Department of Pediatric Hematology and Oncology, U et al. Donor leukocyte infusion after hematopoietic stem cell University Hospital, Motol, Prague, transplantation in patients with juvenile myelomonocytic leukemia. Czech Republic; Leukemia 2005; 19: 971–977. 9Department of Pediatric Hematology, Oncology and 3 Bosi A, Laszlo D, Labopin M, Reffeirs J, Michallet M, Gluckman E Hemostasis, Johann Wolfgang Goethe University Hospital, et al. Second allogeneic bone marrow transplantation in acute Frankfurt am Main, Germany; leukemia: results of a survey by the European Cooperative Group 10Department of Paediatric Haematology/Oncology, for Blood and Marrow Transplantation. J Clin Oncol 2001; 19: University Children’s Hospital, Eberhard-Karls-University, 3675–3684. Tuebingen, Germany; 4 Manabe A, Okamura J, Yumura-Yagi K, Akiyama Y, Sako M, 11Department of Pediatric Hematology and Oncology, Dr von Uchiyama H et al. Allogeneic hematopoietic stem cell transplanta- Haunersches Kinderspital, Children Hospital of the tion for 27 children with juvenile myelomonocytic leukemia Ludwig-Maximilians-University of Munich, Munich, diagnosed based on the criteria of the International JMML working group. Leukemia 2002; 16: 645–649. Germany; 12 5 Yusuf U, Frangoul HA, Gooley TA, Woolfrey AE, Carpenter PA, Department of Pediatrics, University of Kiel, Kiel, Andrews RG et al. Allogeneic bone marrow transplantation in Germany; 13 children with myelodysplastic syndrome or juvenile myelomono- Department of Pediatric Hematology and Oncology, cytic leukemia: the Seattle experience. Bone Marrow Transpl 2004; University Hospital, Giessen, Germany; 33: 805–814. 14 Department of Pediatrics, University of Bologna, S. Orsola 6 Smith FO, King R, Nelson G, Wagner JE, Robertson KA, Sanders JE Hospital, Bologna, Italy; et al. Unrelated donor bone marrow transplantation for children 15Section of Pediatrics, Department of Women’s and with juvenile myelomonocytic leukaemia. Br J Haematol 2002; Children’s Health, Uppsala University, Uppsala, Sweden; 116: 716–724. 16Department of Pediatric Hematology, Our Lady’s Hospital 7 Locatelli F, Niemeyer C, Angelucci E, Bender-Go¨tze C, Burdach S, for Sick Children, Dublin, Ireland; Ebell W et al. Allogenic bone marrow transplantation for chronic 17Dutch Childhood Oncology Group, The Hague, Erasmus myelomonocytic leukemia in childhood: a report from the European University Medical Center-Sophia Children’s Hospital, working group on myelodysplastic syndrome in childhood. J Clin Rotterdam, the Netherlands and Oncol 1997; 15: 566–573. 18Department of Pediatrics, Skejby Hospital, Aarhus 8 Chang YH, Jou ST, Lin DT, Lu MY, Lin KH. Second allogeneic University, Aarhus, Denmark hematopoietic stem cell transplantation for juvenile myelomonocy- E-mail: [email protected] tic 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; sphate (dTMP), dTMP is subsequently phosphorylated to its published online 25 January 2007 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 Acute leukemias are a collection of diseases represented by a rapidly degraded.4 The rate of degradation appears to change in predominance of undifferentiated or immature lymphoid (acute a cell-cycle-dependent manner, resulting in the increased lymphoid leukemia (ALL)) or myeloid (acute myelogenous observed levels of TK1 activity. Cancer cells are known to have leukemia (AML)) precursors in bone marrow and peripheral lost cell-cycle control of TK1, which leads to increased levels of blood. Malignant cells replace normal marrow cells and disrupt TK1 in these cells and could possibly explain the elevations routine blood cell function, leading to death within weeks to found in serum. TK2, the other major TK isozyme, is of months if left untreated. Chemotherapeutic advances have mitochondrial origin and its levels are independent of the cell dramatically altered the management of patients with acute cycle and remain constant in both cancer cells and normal cells, leukemias. Both ALL and AML patients can reasonably expect as well as sera.5 an extended disease-free survival period. Notwithstanding the It has been proposed that serum TK1 levels (i) may indicate improvements made in the treatment of these diseases, some patients’ response to therapy, (ii) may serve as a prognostic patients remain at risk of relapse.1 indicator and (iii) may reflect the aggressiveness of leukemic The ability to monitor patient response to treatment would cells.6 The mechanism of how TK1 enters the blood stream is not enhance disease management. The putative cancer marker, yet fully understood; nevertheless, experimental evidence suggests thymidine kinase (TK), has been shown to be elevated in the that screening serum for elevated TK1 levels may prove to be a serum of patients with a variety of human malignancies.2 TK is a simple and effective means of detecting and monitoring malignant pyrimidine salvage pathway enzyme involved in DNA synthesis disease. The level of serum TK1 in patients with several types of and repair. In the presence of adenosine triphosphate, TK cancer has been shown to correlate with the stage of the disease catalyzes the conversion of thymidine to thymidine monopho- and repeated measurements also reflect relapses and remissions.7

Leukemia Letters to the Editor 561 TK1 has also been shown to be of prognostic value in many solid one-way covariance analysis or Student’s t-test (P40.05) (data tumors, including breast cancer, prostate cancer, bladder carci- not shown). Comparison of the radioassay (data transformed to noma and small-cell carcinoma of the lung.3,6 Serum TK1 levels natural logarithm to normalize the distribution of the data) and correlate strongly with cancer stage and its aggressiveness. In this the immunoassay absorbance data using the Pearson correlation communication, we report that measuring the serum TK1 levels of indicates a strong correlation between TK1 measurements for ALL and AML patients would provide a method for monitoring the assays. The Pearson correlation showed that the correlation patient response to treatment in acute leukemic diseases and was 0.806 (Po0.05), demonstrating that the margin of error is potentially other non-solid tumors. an acceptable value between the radioassay and the immu- This study consists of several phases. In brief, we first noassay. compared the serum TK1 levels of ALL patients with non-cancer Following the encouraging results of the initial phase, these patients. Second, sera from the ALL patients were grouped same 33 patients were analyzed according to the stage of their according to clinical status (pre-treatment, relapse, remission) disease. Figure 2 shows the mean serum TK1 activity that was and analyzed against non-cancer controls. Finally, TK1 levels in determined for the ALL patients who had not yet started sera from individual patients both ALL and AML were treatment, patients who were experiencing relapse and patients sequentially followed for over a year. who were in remission. Relapse patients had a mean serum TK1 In the initial study, TK1 levels were measured in serum activity 82 times higher than the non-cancer control serum TK1 samples from ALL patients (19 females/14 males) and healthy level. These differences were statistically significant (Po0.05). individuals (35 males/14 females; Figure 1). Sera samples from A significant difference (Po0.05) also existed between the both sample groups were measured using a radioassay as well as means of the serum TK1 activity of the pre-treatment ALL an immunoassay employing a TK1-specific monoclonal anti- patients and between the patients who had relapsed after body (mAb) developed in our laboratory.8 treatment. However, no difference (P40.05) existed between Following a natural logarithm transformation to normalize the the control group and the ALL patients currently in remission. distribution of these data, the Student’s t-test and one-way These results indicate the prognostic value of measuring serum ANOVA were performed. The increase in the mean serum TK1 TK1 levels. levels between the non-cancer control individuals and the ALL Having obtained the above results, we decided to monitor patients was statistically significant (Po0.05). Covariance intra-patient TK1 levels. Serial serum samples obtained from analysis was performed and it was determined that gender did nine patients (four ALL and five AML) were collected over a not significantly affect the serum TK1 levels (P40.05). TK1 timeline up to 360 days, and presented as a percentage of the activity was not significantly different in age, blood type, white maximum serum TK1 activity. Serum activity was elevated in blood cell count and race of the ALL patients, as determined by 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– a c). The pattern for patient D (Figure 3d) showed an initial drop in TK1 activity as treatment began, but increased as patient D

4 45

– relapsed into progressive disease. 40 Samples from AML patients (Figure 4) A, B and C were 35 obtained as they began treatment. AML patients D and E had 30 already begun treatment when the initial samples were collected. Results are shown in Figure 4. 25 These patients followed the same trend seen with the ALL 20 patients that were analyzed. The serum TK1 activity of AML 15 patients C, D and E initially decreased (Figure 4c–e). However, 10 all three of these patients relapsed into progressive disease, which corresponded to a substantial increase in serum TK1 5 activity in each patient. TK1 Activity (CPM/mg of protein/hour) x 10 0 Control ALL

b 1.0 4 – 200 n=4 0.8 160 0.6 120 0.4 O.D. 450 80 n=8 0.2 40 n=21 n=49 0

Control ALL TK1 Activity (CPM/mg of protein/hour) x 10 Control Pre- Relapsed Remission Treatment Figure 1 Serum TK1 activity in ALL and non-cancer patients. (a) TK1 activity was measured by radioassay. (b) TK1 levels were detected Figure 2 Serum TK1 activity in pre-treatment, relapsed, remission by immunoassay. (a and b) Data are averages from four separate and control patients. TK1 activity was measured by radioassay. Data experiments (Po0.01). are averages from four separate experiments.

Leukemia Letters to the Editor 562 a 100 b 100 80 80 60 60 40 40

% TK1 Activity of Maximum 20 20 % TK1 Activity of Maximum 0 0 0 50 100 150 200 250 300 350 400 0 20 40 60 80 100 120 140 160 180 Days Days c 100 d 100 80 80 60 60 40 40

% TK1 Activity of Maximum 20 % TK1 Activity of Maximum 20 0 0 0 102030405060708090 0 20 40 60 80 100 120 140 160 Days Days

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.

a 100 b 100 80 80 60 60 40 40 20

% TK1 Activity of Maximum % TK1 Activity of Maximum 20 0 0 0 50 100 150 200 250 300 0 50 100 150 200 250 Days Days

c 100 d 100 80 80 60 60 40 40 20 20 % TK1 Activity of Maximum % TK1 Activity of Maximum 0 0 0 5 10 15 20 25 30 35 40 45 0 1020304050607080 Days Days

e 100 80 60 40

% TK1 Activity of Maximum 20 0 0 50 100 150 200 250 Days

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 each patient who successfully responded to treatment and to increase the chance of survival for the patient. However, went into remission. In each patient that relapsed into there are few tools available for clinicians to monitor progressive disease, the serum TK1 activity began to increase. the progress of the patient during and following treatment. These data suggest that by monitoring serum TK1 activity, These results indicate serum TK1 activity in ALL and AML is the clinician would be able to determine whether or not the an accurate indicator of response to treatment and stage of patient is responding positively to treatment. Long-term disease. The serum TK1 activity dropped significantly in monitoring of the serum TK1 activity would also allow the

Leukemia Letters to the Editor 563 clinician to detect possible relapse in patients if TK1 levels start Acknowledgements to increase. A minor limitation of measuring serum TK1 activity is the We thank KEM Baillie and JM Bridges for providing the serial substantial variance between patients. This variation between blood samples from the ALL and AML patients. We also thank the individuals prevents direct comparison between patients. For Biological Carcinogenesis Branch for providing blood samples. this reason, we presented the changes in serum TK1 activity for the ALL and AML patients as a percentage of the maximum KL O’Neill, F Zhang, H Li, DG Fuja and BK Murray Department of Microbiology and Molecular Biology, measured for each individual (Figures 3 and 4). Our results show Brigham Young University, Provo, UT, USA that the changes in serum TK1 activity are only useful for E-mail: [email protected] prognosis when compared with the individual’s previous samples. Therefore, a baseline level of serum TK1 activity must References be established for each individual if serum TK1 is to be used as a prognostic tool in leukemia, similar to the method currently 1 Kantarjian HM. Adult acute lymphocytic leukemia: critical review used in monitoring prostate-specific antigen in men. Variations of current knowledge. Am J Med 1994; 97: 176–184. in TK1 levels between serial samples from the same individual 2 O’Neill KL, Buckwalter MR, Murray BK. Thymidine kinase: are directly reflective of the response to treatment, indicating diagnostic and prognostic potential. Expert Rev Mol Diagn 2001; that TK1 measurements can accurately predict patient prog- 1: 428–433. nosis. 3 Hannigan BM, Barnett YA, Armstrong DB, McKelvey-Martin VJ, McKenna PG. Thymidine kinases: the enzymes and their clinical Our results comparing the mean serum TK1 levels of ALL usefulness. Cancer Biother 1993; 8: 189–197. patients with healthy individuals indicated statistically 4 Sherley JL, Kelly TJ. Regulation of human thymidine kinase during significant differences between the two groups. This confirms the cell cycle. J Biol Chem 1988; 263: 8350–8358. the reality of measuring serum TK1 levels as a diagnostic 5 Johansson M, Karlsson A. Cloning of the cDNA and chromosome tool. Statistical significance between serum TK1 levels in ALL localization of the gene for human thymidine kinase 2. J Biol Chem patients of different stages confirms TK1 as a possible prognostic 1997; 272: 8454–8458. 6 Zhang J, Jia Q, Zou S, Zhang P, Zhang X, Skog S et al. Thymidine tool. kinase 1: a proliferation marker for determining prognosis and This study also indicates that the immunoassay, utilizing monitoring the surgical outcome of primary bladder carcinoma the TK1-specific mAb, is an accurate method of measuring patients. Oncol Rep 2006; 15: 455–461. serum TK1 levels. The development of an immunoassay 7 He Q, Zhang P, Zou L, Li H, Wang X, Zhou S et al. Concentration of to measure serum TK1 levels would be more practical and thymidine kinase 1 in serum (S-TK1) is a more sensitive proliferation efficient for use in a clinical setting. Through continued marker in human solid tumors than its activity. Oncol Rep 2005; 14: 1013–1019. investigation of TK1, we hope to provide clinicians with a 8 Zhang F, Shao X, Li H, Robison JG, Murray BK, O’Neill KL. A valuable tool for earlier detection and improved evaluation of monoclonal antibody specific for human thymidine kinase 1. treatment success. 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; phorylation of STAT5 in M0-91, cell lysates were trypsin- published online 25 January 2007 digested, 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 The TEL-TRKC gene fusion associated with the t(12;15)(p13;q25) 393 phosphotyrosine sites in 265 proteins (Supplementary translocation has been implicated in both hematological Table 1). Among these proteins, over 15 tyrosine kinases (acute myeloid leukemia (AML))1 and non-hematological were tyrosine-phosphorylated (Figure 1b). Multiple tyrosine- malignancies (congenital fibrosarcoma, congenital mesoblastic phosphorylated peptides corresponded to either TRKB or TRKC, nephroma and secretory breast carcinoma). In AML, the including three tyrosines in the activation loop. Thus, either TEL-TRKC (TEL-TRKC(L)) derives from the in-frame fusion of TRKB or TRKC could be aberrantly activated in M0-91 exons 1–4 of TEL to exons 13–18 of TRKC. In contrast, the cells. While full-length TRKB/C have a molecular weight of TEL-TRKC variant associated with solid tumors (TEL-TRKC(F)) 140–145 kDa, we observed a 50 kDa by Western blot with a contains exons 1–5 of the TEL gene. Activation of both the pan-TRK antibody in M0-91 cells (Figure 1c). In addition, we RAS-MAPK and PI3K-AKT pathways by TEL-TRKC contributes observed tyrosine-phosphorylated peptides deriving from the to oncogenic signaling in transfected NIH3T3 cells.2 However, TEL protein (Supplementary Table 1). TEL is a member of the ETS no human cell lines are available to study the TEL-TRKC family transcription factor and is essential for hematopoiesis. It is fusion. a frequent target of chromosomal translocations in human In this study, we screened over 40 AML cell lines for cancers, and one of its fusion partners is TRKC.1 To determine constitutive phosphorylation of STAT5 by Western blot. The whether a chimeric TEL-TRKC transcript was present, we M0-91, an AML-M0-derived cell line3, showed constitutive performed 30 rapid amplification of complementary DNA tyrosine phosphorylation of STAT5 (Figure 1a). To identify (cDNA) ends on the sequence encoding the HLH domain of protein tyrosine kinases responsible for the constitutive phos- TEL. Sequence analysis of the resultant product revealed that the

Leukemia