Letters to the Editor 1191 in MM in two distinct poor prognosis subsets and have initiated 4First Department of Medicine, Center for Oncology and Hematology, the phase of NGS work that will be necessary to elucidate at the Wilhelminenspital, Vienna, Austria and gene level the ‘broad-brush’ insight delivered by the study by 5The GenePool, Ashworth Laboratories, University of Edinburgh, Keats et al.8 Taken together, our observations and those from the Edinburgh, UK single t(4;14) case9 reveal a disease landscape at relapse in MM E-mail: [email protected] that displays complex patterns of genetic mutations that will need 6Joint first authors in this study. 7 to be dissected by NGS in relation to specific therapies. Common Joint senior authors in this study. mutations, such as CSMD3 and SUB1 as described above, may provide key pointers to essential survival pathways irrespective of type of therapy or disease subset. Whether specific patterns associate with individual markers of poor prognosis in MM will REFERENCES emerge from NGS studies of larger cohorts. These patterns will 1 Carrasco DR, Tonon G, Huang Y, Zhang Y, Sinha R, Feng B et al. High-resolution define the models of clonal evolution, which may be linear or may genomic profiles define distinct clinico-pathogenetic subgroups of multiple involve a clone regressing and reappearing by competition. myeloma patients. Cancer Cell 2006; 9: 313–325. 2 Chesi M, Bergsagel PL. Many multiple myelomas: making more of the molecular Whether all 1q21 MM cases exhibit an apparent linear mode of mayhem. Hematology Am Soc Hematol Educ Program 2011; 2011: 344–353. evolution under therapy, as suggested by our data, remains to be 3 Hanamura I, Stewart JP, Huang Y, Zhan F, Santra M, Sawyer JR et al. Frequent gain determined. of chromosome band 1q21 in plasma-cell dyscrasias detected by fluorescence It is however evident from these two initial NGS studies that the in situ hybridization: incidence increases from MGUS to relapsed myeloma and is tumor clone will need to be closely scrutinized after each phase of related to prognosis and disease progression following tandem stem-cell trans- treatment. These studies also suggest that a multi-target plantation. Blood 2006; 108: 1724–1732. combination therapy may be required to eradicate the genetically 4 Klein U, Jauch A, Hielscher T, Hillengrass J, Raab MS, Seckinger A et al. variant subclones in any specific tumor population in MM that Chromosomal aberrations þ 1q21 and del(17p13) predict survival in patients with persist after early therapy to prevent escape.10 recurrent multiple myeloma treated with lenalidomide and dexamethasone. Cancer 2011; 117: 2136–2144. 5 Kumar SK, Mikhael JR, Buadi FK, Dingli D, Fonseca R, Gertz MA et al. Management CONFLICT OF INTEREST of newly diagnosed symptomatic multiple myeloma: updated mayo Stratification of Myeloma and Risk-Adapted Therapy (mSMART) consensus guidelines. Mayo The authors declare no conflict of interest. Clinic Proc 2009; 84: 1095–1110. 6 Chapman MA, Lawrence MS, Keats JJ, Cibulskis K, Sougnez C, Schinzel AC et al. ACKNOWLEDGEMENTS Initial genome sequencing and analysis of multiple myeloma. Nature 2011; 471: 467–472. This work was funded by EU FP7 Program Project 278706 ‘OVER-MyR’, Leukemia & 7 Walker BA, Wardell CP, Melchor L, Hulkki S, Potter NE, Johnson DC et al. Intraclonal Lymphoma Research (UK) and Cancer Research UK (JG). heterogeneity and distinct molecular mechanisms characterize the development of t(4;14) and t(11;14) myeloma. Blood 2012; 120: 1077–1086. 8 Keats JJ, Chesi M, Egan JB, Garbitt VM, Palmer SE, Braggio E et al. Clonal compe- 1,6 2,6 3 2 4 N Weston-Bell , J Gibson , M John , S Ennis , S Pfeifer , tition with alternating dominance in multiple myeloma. Blood 2012; 120: T Cezard5, H Ludwig4, A Collins2,7, N Zojer4,7 and SS Sahota1,7 1067–1076. 1Tumour Immunogenetics Group, Cancer Sciences Academic Unit, 9 Egan JB, Shi CX, Tembe W, Christoforides A, Kurdoglu A, Sinari S et al. Whole Faculty of Medicine, University of Southampton, Southampton, UK; genome sequencing of multiple myeloma from diagnosis to plasma cell leukemia 2Genetic Epidemiology and Genomic Informatics Group, reveals genomic initiating events, evolution and clonal tides. Blood 2012; 120: Human Development and Health Academic Unit, 1060–1066. 10 Al-Lazikani B, Banerji U, Workman P. Combinatorial drug therapy for cancer in the Faculty of Medicine, University of Southampton, post-genomic era. Nat Biotechnol 2012; 30: 679–692. Southampton, UK; 11 Fuentes Fajardo KV, Adams D, Mason CE, Sincan M, Tifft C. NISC Comparative 3 Department of Preclinical Sciences, Faculty of Medical Sciences, Sequencing Program. Detecting false-positive signals in exome sequencing. Hum University of The West Indies, Trinidad and Tobago; Mutat 2012; 33: 609–613.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Matriptase is highly upregulated in chronic lymphocytic leukemia and promotes cancer cell invasion

Leukemia (2013) 27, 1191–1194; doi:10.1038/leu.2012.289 Studies indicate that matriptase may have an important role in cancer. Matriptase overexpression has been reported in many human solid tumors, including prostate, breast, Matriptase, also called MT-SP1 or epithin, is a type-II transmem- ovarian, kidney and lung cancers.4,5 In mice, transgenic brane serine protease expressed on the surface of the normal matriptase expression in keratinocytes causes skin carcinoma.6 epithelium.1,2 The enzyme is required for epidermal differentiation The molecular mechanism under which matriptase may and barrier function in the skin. Low levels of matriptase mRNA participate in cancer is not fully understood. Matriptase has also were detected in human blood cells including monocytes and been shown to activate pro-urokinase, prostasin, protease-acti- B lymphocytes,3 but the biological significance of such expression vated receptor-2, pro-hepatocyte growth factor, pro-macrophage is unclear. In monocytes, matriptase may be involved in initiating stimulating protein-1, platelet-derived growth factor, vascular plasminogen activation.3 endothelial growth factor receptor 2 and Tie2 receptor.2,4,7–9 It is

Accepted article preview online 9 October 2012; advance online publication, 26 October 2012

& 2013 Macmillan Publishers Limited Leukemia (2013) 1172 – 1218 Letters to the Editor 1192

Figure 1. Matriptase expression in hematological cancer cell lines and bone marrow samples from leukemia patients. Matriptase mRNA (a) and protein (b) expression was detected in Namalwa and Raji cells by RT-PCR and western blotting. Matriptase mRNA (c) and protein (d) expression in bone marrow cells from leukemia patients. Data are representative of 223 patient samples. (e) Matriptase protein levels in selected western blots analyzed by densitometry. Sample numbers in each group are indicated. (f) Matriptase and CD19 immunostaining in Namalwa, CLL and NPB cells. Nuclei are labeled in blue. Data are representative of X3 experiments. Bar: 10 mm. (g) Flow cytometric analysis of matriptase (top panels) and CD19 (lower panels) expression in NPB, Namalwa and CLL cells. Percentages of positive cells are indicated. Data are representative of X3 experiments.

possible that these matriptase activities may promote cancer detected matriptase protein in most CLL samples (79/81, 97.5%) growth and invasion. but few samples of ALL (2/74, 2.7%) and AML (2/56, 3.6%) and In this study, we tested the hypothesis that matripase may also none in CML (n ¼ 12) or normal peripheral blood (NPB) samples participate in hematological cancers. By reverse transcriptase PCR (n ¼ 10). Representative western blots (Figure 1d) and quantitative (RT-PCR), we screened matriptase mRNA expression in hematolo- analysis of selected western blots (Figure 1e) are shown. Similar gical cancer cell lines and bone marrow cells from leukemia results also were found by quantitative PCR (qPCR), which showed patients. Matriptase mRNA was detected in B-cell-derived 420-fold increases of matriptase mRNA expression in CLL Namalwa and Raji cells, but not in T-cell-derived (Hs 505.T, samples compared with normal controls or other types of MOLT4, CCRF-CEM, Jurkat), chronic myeloid leukemia (CML)- leukemia (Supplementary Figure 1). derived (KU-812, MEG-01, K562) or acute myeloid leukemia (AML)- Matriptase is a transmembrane protein.1 By immunostaining derived (U937, HEL, SH-2, SHI-1, THP-1, NB4) cells (Figure 1a). A under non-permeable conditions, strong matriptase staining was similar matriptase protein expression pattern in these cell lines was detected on the surface of Namalwa and CLL cells but not NPB confirmed by western blotting (Figure 1b), suggesting that cells (Figure 1f). A similar staining pattern was found with matriptase may be upregulated in B-cell-derived cancers. CD19, an established B-cell surface marker (Figure 1f). In contrast, We then examined matriptase expression in bone marrow positive staining for CD3, a T-cell surface marker, was detected in samples from 223 patients with different types of leukemia, NPB cells but not Namalwa and CLL cells (Supplementary including chronic lymphocytic leukemia (CLL) (n ¼ 81), acute Figure 2). We confirmed matriptase cell surface expression by lymphocytic leukemia (ALL) (n ¼ 74), AML (n ¼ 56) and CML flow cytometry, which showed that 490% of Namalwa and CLL (n ¼ 12). The study was approved by the ethics committee of cells were matriptase positive, whereas only B1% NPB cells were Soochow University and all patients and normal controls (n ¼ 10) matriptase positive (Figure 1g). Similar results were found for gave written informed consent. By RT-PCR, matriptase mRNA was CD19 cell surface expression (Figure 1g; Supplementary Figure 3). detected in the majority of CLL samples (72/74, 97.3%), whereas These data indicate that matriptase is present on the CLL cell most ALL, AML and CML samples were negative or weakly positive surface and may serve as a new cell surface marker for these (Figure 1c). Similar results were found by western blotting, which leukemia cells.

Leukemia (2013) 1172 – 1218 & 2013 Macmillan Publishers Limited Letters to the Editor 1193

Figure 2. Matriptase inhibition reduced cancer cell invasion in matrigels. Matriptase mRNA (a) and protein (b) levels were reduced in Namalwa cells transduced with matriptase-specific shRNA (siM) compared with control shRNA (siC)-transduced cells. Matrigel invasion (c), but not cell proliferation measured by a colorimetric assay (d), was markedly reduced in siM-transduced Namalwa cells. Recombinant HAI-1-KD1 inhibited Namalwa cell invasion (e) but not migration (f) or proliferation (g). Soybean trypsin inhibitor (STI) was used as a negative control. HAI-1-KD1 inhibited the invasion of Raji (h) and CLL (i), but not CML (j) cells. Data are mean±s.d. from X3 experiments.

The activity of matriptase is controlled by its cognate inhibitors, matriptase, on cancer cell invasion. HAI-1-KD1, but not control hepatocyte growth factor activator inhibitor (HAI)-1 and -2.1,4,10 Kunitz-type soybean trypsin inhibitor, dose dependently inhibited Uncontrolled matriptase activity has been implicated in cancer Namalwa and Raji cells and bone marrow cells from CLL patients development and progression. By qPCR, we did not detect HAI-1 in matrigel invasion (Figures 2e, h and i). Under these conditions, and HAI-2 mRNA upregulation in bone marrow cells from CLL HAI-1-KD1 did not inhibit Namalwa, Raji or CLL cell proliferation or patients (Supplementary Figure 4). Instead, HAI-1 and HAI-2 mRNA transwell migration in the absence of matrigels (Figures 2f and g) upregulation was found in both B- and T-ALL cells, which were (Supplementary Figures 5A and B). In another experiment, mostly matriptase negative (Supplementary Figure 4). The data HAI-1-KD1 did not inhibit matriptase-negative bone marrow cells indicated that, unlike in many solid tumors,11,12 matriptase from CML patients in matrigel invasion (Figure 2j) or migration in expression in CLL was not coupled with HAI-1 or HAI-2 the absence of matrigels (Supplementary Figure 5C). expression, suggesting that increased matriptase activity may In summary, we report for the first time that matriptase is highly not be inhibited in CLL cells. expressed in CLL cells. As matriptase is also expressed in Namalwa To examine the potential role of matriptase in cancer invasion, and Raji cells (Figure 1), which are of Burkitt lymphoma origin, we used small hairpin (sh) RNA to silence matriptase expression in matriptase is probably expressed in other B-cell non-Hodgkin Namalwa cells. By qPCR and western blotting, matriptase mRNA lymphomas. A preliminary study indeed detected matriptase and protein levels were decreased 490% in cells transduced expression in some patient samples of follicular lymphoma, with matripase-specific shRNA (siM) compared with that in marginal zone lymphoma and diffuse large B-cell lymphoma cells transduced with a non-silencing shRNA control (siC) (data not shown), supporting that matriptase may be expressed in (Figures 2a and b). In a transwell assay, cell invasion across B-cell-derived malignancies. Interestingly, matriptase expression matrigels was reduced by 89.1±5.9% in siM-transduced cells was not observed in AML, CML, T-ALL and B-ALL cells (Figure 1 compared with that in control cells (Figure 2c). In these cells, and Supplementary Figure 1). Thus, among leukemia cells, matrip- inhibition of matriptase expression did not appear to alter cell tase appeared to be expressed mostly in CLL cells, suggesting that proliferation, as measured by a colorimetric assay (Figure 2d). matriptase may be used as a new cell surface marker for CLL. We next examined the effect of a recombinant HAI-1 fragment Proteolytic enzymes are known to have important roles in containing Kunitz domain 1 (HAI-1-KD1),13 which inhibits cancers.14,15 We showed that inhibition of matriptase by either

& 2013 Macmillan Publishers Limited Leukemia (2013) 1172 – 1218 Letters to the Editor 1194 shRNA silencing or a recombinant matriptase inhibitor blocked 6Molecular Cardiology, Nephrology and Hypertension, Namalwa and Raji cells and patient CLL cells in matrigel invasion Cleveland Clinic, Cleveland, OH, USA (Figure 2). The results indicate that matriptase may contribute to E-mail: [email protected] the invasiveness of these B-cell-derived cancer cells, either directly or E-mail: [email protected] by degrading matrix proteins or indirectly by activating growth 7These authors contributed equally to this work. factors or other unknown mechanisms. Our findings suggest that matriptase inhibition may be tested as a new therapeutic strategy to prevent CLL cell invasion and metastasis. REFERENCES 1 Lin CY, Tseng IC, Chou FP, Su SF, Chen YW, Johnson MD et al. Zymogen activation, inhibition, and ectodomain shedding of matriptase. Front Biosci 2008; 13: 621–635. CONFLICT OF INTEREST 2 Szabo R, Bugge TH. Membrane-anchored serine proteases in vertebrate cell and developmental biology. Annu Rev Cell Dev Biol 2011; 27: 213–235. The authors declare no conflict of interest. 3 Kilpatrick LM, Harris RL, Owen KA, Bass R, Ghorayeb C, Bar-Or A et al. Initiation of plasminogen activation on the surface of monocytes expressing the type II ACKNOWLEDGEMENTS transmembrane serine protease matriptase. Blood 2006; 108: 2616–2623. 4 Darragh MR, Bhatt AS, Craik CS. MT-SP1 proteolysis and regulation of This study was supported in part by grants from the National Natural Science cell-microenvironment interactions. Front Biosci 2008; 13: 528–539. Foundation of China (31070716, 81170247 and 31161130356) and the Priority 5 List K. Matriptase: a culprit in cancer? Future Oncol 2009; 5: 97–104. Academic Program Development of Jiangsu Higher Education Institutions. 6 List K, Szabo R, Molinolo A, Sriuranpong V, Redeye V, Murdock T et al. Deregulated matriptase causes ras-independent multistage carcinogenesis and promotes ras-mediated malignant transformation. Genes Dev 2005; 19: 1934–1950. AUTHOR CONTRIBUTIONS 7 Kim C, Lee HS, Lee D, Lee SD, Cho EG, Yang SJ et al. Epithin/PRSS14 proteolytically LG, ML and ND designed and performed experiments and wrote regulates angiopoietin receptor Tie2 during transendothelial migration. Blood the paper. YJ performed experiments. C-YL and MH provided key reagents. 2011; 117: 1415–1424. 8 Lee SL, Dickson RB, Lin CY. Activation of hepatocyte growth factor and urokinase/ DW provided clinical samples. QW designed experiments and wrote plasminogen activator by matriptase, an epithelial membrane serine protease. the paper; all authors participated in discussions and critically read the J Biol Chem 2000; 275: 36720–36725. manuscript. 9 Ustach CV, Huang W, Conley-LaComb MK, Lin CY, Che M, Abrams J et al. A novel signaling axis of matriptase/PDGF-D/ss-PDGFR in human prostate cancer. L Gao1,7, M Liu1,7, N Dong1,2, Y Jiang1, C-Y Lin3, M Huang4,5, Cancer Res 2010; 70: 9631–9640. DWu2 and Q Wu1,5,6 10 Bugge TH, Antalis TM, Wu Q. Type II transmembrane serine proteases. J Biol Chem 1 2009; 284: 23177–23181. The Cyrus Tang Hematology Center, Jiangsu Institute of 11 Baba T, Kawaguchi M, Fukushima T, Sato Y, Orikawa H, Yorita K et al. Loss of Hematology, the First Affiliated Hospital, membrane-bound serine protease inhibitor HAI-1 induces oral squamous cell Soochow University, Suzhou, China; carcinoma cells invasiveness. J Pathol 2012; 228: 181–192. 2 Thrombosis and Hemostasis Key Laboratory of the Ministry of 12 Xu H, Xu Z, Tseng IC, Chou FP, Chen YW, Wang JK et al. Mechanisms for the Health, Jiangsu Institute of Hematology, The First Affiliated Hospital, control of matriptase activity in the absence of sufficient HAI-1. Am J Physiol Cell Soochow University, Suzhou, China; Physiol 2012; 302: C453–C462. 3Lombardi Comprehensive Cancer Center, Georgetown University, 13 Kirchhofer D, Peek M, Li W, Stamos J, Eigenbrot C, Kadkhodayan S et al. Tissue School of Medicine, Washington, DC, USA; expression, protease specificity, and Kunitz domain functions of hepatocyte growth 4State Key Laboratory of Structural Chemistry, Fujian Institute of factor activator inhibitor-1B (HAI-1B), a new splice variant of HAI-1. JBiolChem2003; 278: 36341–36349. Research on the Structure of Matter, Chinese Academy of 14 Egeblad M, Werb Z. New functions for the matrix metalloproteinases in cancer Sciences, Fuzhou, China; progression. Nat Rev Cancer 2002; 2: 161–174. 5 Danish-Chinese Center of Proteases and Cancer, 15 Lopez-Otin C, Matrisian LM. Emerging roles of proteases in tumour suppression. Cleveland, OH, USA and Nat Rev Cancer 2007; 7: 800–808.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Evaluation of WT1 expression in bone marrow vs peripheral blood samples of children with acute myeloid leukemia—impact on minimal residual disease detection

Leukemia (2013) 27, 1194–1196; doi:10.1038/leu.2012.291 In this report, we focus on the role of WT1 as a marker for monitoring of malignant cells in patients with childhood acute myeloid leukemia (AML). Many important studies proved the Wilms’ tumor gene 1 (WT1) is located on chromosome 11p13. As a usefulness of WT1 for this purpose, showing that this gene provides zinc finger transcription factor, WT1 regulates expression of many a promising tool for residual disease detection. In general, these target genes involved in regulation of cell cycle, proliferation, studies were based on the analysis of bone marrow (BM) samples,6–8 differentiation and apoptosis.1,2 Overexpression of WT1 was while only minority used peripheral blood (PB) as a source of leukemic foundinmajorityofacuteleukemias and other hematological cells.9,10 Considering the fact, that the percentage of blasts can differ malignancies. WT1 has, therefore, been intensively studied as a between BM and PB, one could assume that the choice of material for potential prognostic factor, marker for minimal residual disease WT1 detection may influence the subsequent analysis. Nevertheless, (MRD) and target for immunotherapy (summarized in references1,3–5). no consensus has been made so far.

Accepted article preview online 10 October 2012; advance online publication, 13 November 2012

Leukemia (2013) 1172 – 1218 & 2013 Macmillan Publishers Limited