Genetic mutations in acute myeloid leukaemia (AML) are assumed to occur in a sequential order; however, the predominant hierarchical roles of specific mutated genes have not been fully described. In this study, we aimed to determine the clonal involvement of the most frequent AML-associated mutations. Using a targeted sequencing panel for 18 genes, we traced changes and relative clonal contribution of mutations in 52 patients. We analysed 35 pairs of diagnosis and relapse samples, 27 pairs of primary samples and corresponding patient-derived xenografts, and 34 pairs of total leukocytes and corresponding isolated primitive cells or blast populations. In both relapse and xenografts, we observed conservation of main leukaemic clones and variability was limited to subclones with late-acquired mutations. AML evolution thus mainly involved modification of subclones while the clonal background remained unchanged. NPM1 mutations were identified as the most probable leukaemia-transformation lesion, remaining conserved in contrast to high variation of accompanying subclonal FLT3 and NRAS mutations. DNMT3A mutations represented the most stable mutations forming a preleukaemic background in most samples. Mutations in genes IDH1/2, TET2, RUNX1, ASXL1 and U2AF1 were detected both as preleukaemic and as subclonal lesions, suggesting a non-specific order of acquisition.

Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Internal Medicine Hematology and Oncology Faculty of Medicine Masaryk University Brno Czech Republic

Department of Internal Medicine Hematology and Oncology University Hospital Brno Brno Czech Republic

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Cancer Genome Atlas Research Network (2013) Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. New England Journal of Medicine, 368, 2059-2074.

Chen, J., Kao, Y.R., Sun, D., Todorova, T.I., Reynolds, D., Narayanagari, S.R., Montagna, C., Will, B., Verma, A. & Steidl, U. (2019) Myelodysplastic syndrome progression to acute myeloid leukemia at the stem cell level. Nature Medicine, 25, 103-110.

Corces-Zimmerman, M.R., Hong, W.-J., Weissman, I.L., Medeiros, B.C. & Majeti, R. (2014) Preleukemic mutations in human acute myeloid leukemia affect epigenetic regulators and persist in remission. Proceedings of the National Academy of Sciences of the United States of America, 111, 2548-2553.

Culen, M., Kosarova, Z., Jeziskova, I., Folta, A., Chovancova, J., Loja, T., Tom, N., Bystry, V., Janeckova, V., Dvorakova, D., Mayer, J. & Racil, Z. (2018) The influence of mutational status and biological characteristics of acute myeloid leukemia on xenotransplantation outcomes in NOD SCID gamma mice. Journal of Cancer Research and Clinical Oncology, 144, 1239-1251.

Ding, L., Ley, T.J., Larson, D.E., Miller, C.A., Koboldt, D.C., Welch, J.S., Ritchey, J.K., Young, M.A., Lamprecht, T., McLellan, M.D., McMichael, J.F., Wallis, J.W., Lu, C., Shen, D., Harris, C.C., Dooling, D.J., Fulton, R.S., Fulton, L.L., Chen, K., Schmidt, H., Kalicki-Veizer, J., Magrini, V.J., Cook, L., McGrath, S.D., Vickery, T.L., Wendl, M.C., Heath, S., Watson, M.A., Link, D.C., Tomasson, M.H., Shannon, W.D., Payton, J.E., Kulkarni, S., Westervelt, P., Walter, M.J., Graubert, T.A., Mardis, E.R., Wilson, R.K. & DiPersio, J.F. (2012) Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing. Nature, 481, 506-510.

Dohner, H., Estey, E.H., Amadori, S., Appelbaum, F.R., Buchner, T., Burnett, A.K., Dombret, H., Fenaux, P., Grimwade, D., Larson, R.A., Lo-Coco, F., Naoe, T., Niederwieser, D., Ossenkoppele, G.J., Sanz, M.A., Sierra, J., Tallman, M.S., Lowenberg, B. & Bloomfield, C.D. (2010) Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood, 115, 453-474.

Dohner, H., Estey, E., Grimwade, D., Amadori, S., Appelbaum, F.R., Buchner, T., Dombret, H., Ebert, B.L., Fenaux, P., Larson, R.A., Levine, R.L., Lo-Coco, F., Naoe, T., Niederwieser, D., Ossenkoppele, G.J., Sanz, M., Sierra, J., Tallman, M.S., Tien, H.-F., Wei, A.H., Lowenberg, B. & Bloomfield, C.D. (2017) Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood, 129, 424-447.

Genovese, G., Kahler, A.K., Handsaker, R.E., Lindberg, J., Rose, S.A., Bakhoum, S.F., Chambert, K., Mick, E., Neale, B.M., Fromer, M., Purcell, S.M., Svantesson, O., Landen, M., Hoglund, M., Lehmann, S., Gabriel, S.B., Moran, J.L., Lander, E.S., Sullivan, P.F., Sklar, P., Gronberg, H., Hultman, C.M. & McCarroll, S.A. (2014) Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence. New England Journal of Medicine, 371, 2477-2487.

Grove, C.S. & Vassiliou, G.S. (2014) Acute myeloid leukaemia: a paradigm for the clonal evolution of cancer? Disease Models & Mechanisms, 7, 941-951.

Haferlach, C., Kohlmann, A., Kern, W., Haferlach, T. & Schnittger, S. (2013) Cytogenetic and molecular genetic characterization of MLL-PTD positive AML in comparison to MLL-translocated AML [abstract]. Blood, 122, 2557a.

Hirsch, P., Zhang, Y., Tang, R., Joulin, V., Boutroux, H., Pronier, E., Moatti, H., Flandrin, P., Marzac, C., Bories, D., Fava, F., Mokrani, H., Betems, A., Lorre, F., Favier, R., Feger, F., Mohty, M., Douay, L., Legrand, O., Bilhou-Nabera, C., Louache, F. & Delhommeau, F. (2016) Genetic hierarchy and temporal variegation in the clonal history of acute myeloid leukaemia. Nature Communications, 7, 12475.

Höllein, A., Meggendorfer, M., Dicker, F., Jeromin, S., Nadarajah, N., Kern, W., Haferlach, C. & Haferlach, T. (2018) NPM1 mutated AML can relapse with wild-type NPM1: persistent clonal hematopoiesis can drive relapse. Blood Advances, 2, 3118-3125.

Jaiswal, S., Fontanillas, P., Flannick, J., Manning, A., Grauman, P.V., Mar, B.G., Lindsley, R.C., Mermel, C.H., Burtt, N., Chavez, A., Higgins, J.M., Moltchanov, V., Kuo, F.C., Kluk, M.J., Henderson, B., Kinnunen, L., Koistinen, H.A., Ladenvall, C., Getz, G., Correa, A., Banahan, B.F., Gabriel, S., Kathiresan, S., Stringham, H.M., McCarthy, M.I., Boehnke, M., Tuomilehto, J., Haiman, C., Groop, L., Atzmon, G., Wilson, J.G., Neuberg, D., Altshuler, D. & Ebert, B.L. (2014) Age-related clonal hematopoiesis associated with adverse outcomes. New England Journal of Medicine, 371, 2488-2498.

Jan, M., Snyder, T.M., Corces-Zimmerman, M.R., Vyas, P., Weissman, I.L., Quake, S.R. & Majeti, R. (2012) Clonal evolution of preleukemic hematopoietic stem cells precedes human acute myeloid leukemia. Science Translational Medicine, 4, 149ra118.

Klco, J.M., Spencer, D.H., Miller, C.A., Griffith, M., Lamprecht, T.L., O'Laughlin, M., Fronick, C., Magrini, V., Demeter, R.T., Fulton, R.S., Eades, W.C., Link, D.C., Graubert, T.A., Walter, M.J., Mardis, E.R., Dipersio, J.F., Wilson, R.K. & Ley, T.J. (2014) Functional heterogeneity of genetically defined subclones in acute myeloid leukemia. Cancer Cell, 25, 379-392.

Koboldt, D.C., Chen, K., Wylie, T., Larson, D.E., McLellan, M.D., Mardis, E.R., Weinstock, G.M., Wilson, R.K. & Ding, L. (2009) VarScan: variant detection in massively parallel sequencing of individual and pooled samples. Bioinformatics, 25, 2283-2285.

Koboldt, D.C., Zhang, Q., Larson, D.E., Shen, D., McLellan, M.D., Lin, L., Miller, C.A., Mardis, E.R., Ding, L. & Wilson, R.K. (2012) VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Research, 22, 568-576.

Kronke, J., Bullinger, L., Teleanu, V., Tschurtz, F., Gaidzik, V.I., Kuhn, M.W., Rucker, F.G., Holzmann, K., Paschka, P., Kapp-Schworer, S., Spath, D., Kindler, T., Schittenhelm, M., Krauter, J., Ganser, A., Gohring, G., Schlegelberger, B., Schlenk, R.F., Dohner, H. & Dohner, K. (2013) Clonal evolution in relapsed NPM1-mutated acute myeloid leukemia. Blood, 122, 100-108.

Lai, Z., Markovets, A., Ahdesmaki, M., Chapman, B., Hofmann, O., McEwen, R., Johnson, J., Dougherty, B., Barrett, J.C. & Dry, J.R. (2016) VarDict: a novel and versatile variant caller for next-generation sequencing in cancer research. Nucleic Acids Research, 44, e108.

Metzeler, K.H., Herold, T., Rothenberg-Thurley, M., Amler, S., Sauerland, M.C., Görlich, D., Schneider, S., Konstandin, N.P., Dufour, A., Bräundl, K., Ksienzyk, B., Zellmeier, E., Hartmann, L., Greif, P.A., Fiegl, M., Subklewe, M., Bohlander, S.K., Krug, U., Faldum, A., Berdel, W.E., Wörmann, B., Büchner, T., Hiddemann, W., Braess, J. & Spiekermann, K. (2016) Spectrum and prognostic relevance of driver gene mutations in acute myeloid leukemia. Blood, 128, 686-698.

Papaemmanuil, E., Gerstung, M., Bullinger, L., Gaidzik, V.I., Paschka, P., Roberts, N.D., Potter, N.E., Heuser, M., Thol, F., Bolli, N., Gundem, G., Van Loo, P., Martincorena, I., Ganly, P., Mudie, L., McLaren, S., O'Meara, S., Raine, K., Jones, D.R., Teague, J.W., Butler, A.P., Greaves, M.F., Ganser, A., Dohner, K., Schlenk, R.F., Dohner, H. & Campbell, P.J. (2016) Genomic Classification and Prognosis in Acute Myeloid Leukemia. New England Journal of Medicine, 374, 2209-2221.

Potter, N., Miraki-Moud, F., Ermini, L., Titley, I., Vijayaraghavan, G., Papaemmanuil, E., Campbell, P., Gribben, J., Taussig, D. & Greaves, M. (2019) Single cell analysis of clonal architecture in acute myeloid leukaemia. Leukemia, 33, 1113-1123.

Shlush, L.I., Zandi, S., Mitchell, A., Chen, W.C., Brandwein, J.M., Gupta, V., Kennedy, J.A., Schimmer, A.D., Schuh, A.C., Yee, K.W., McLeod, J.L., Doedens, M., Medeiros, J.J.F., Marke, R., Kim, H.J., Lee, K., McPherson, J.D., Hudson, T.J., The HALT Pan-Leukemia Gene Panel Consortium, Brown, A.M., Yousif, F., Trinh, Q.M., Stein, L.D., Minden, M.D., Wang, J.C.Y. & Dick, J.E. (2014) Identification of preleukaemic haematopoietic stem cells in acute leukaemia. Nature, 506, 328-333.

Sun, Q.Y., Ding, L.W., Tan, K.T., Chien, W., Mayakonda, A., Lin, D.C., Loh, X.Y., Xiao, J.F., Meggendorfer, M., Alpermann, T., Garg, M., Lim, S.L., Madan, V., Hattori, N., Nagata, Y., Miyano, S., Yeoh, A.E., Hou, H.A., Jiang, Y.Y., Takao, S., Liu, L.Z., Tan, S.Z., Lill, M., Hayashi, M., Kinoshita, A., Kantarjian, H.M., Kornblau, S.M., Ogawa, S., Haferlach, T., Yang, H. & Koeffler, H.P. (2017) Ordering of mutations in acute myeloid leukemia with partial tandem duplication of MLL (MLL-PTD). Leukemia, 31, 1-10.

Thol, F., Klesse, S., Kohler, L., Gabdoulline, R., Kloos, A., Liebich, A., Wichmann, M., Chaturvedi, A., Fabisch, J., Gaidzik, V.I., Paschka, P., Bullinger, L., Bug, G., Serve, H., Gohring, G., Schlegelberger, B., Lubbert, M., Kirchner, H., Wattad, M., Kraemer, D., Hertenstein, B., Heil, G., Fiedler, W., Krauter, J., Schlenk, R.F., Dohner, K., Dohner, H., Ganser, A. & Heuser, M. (2017) Acute myeloid leukemia derived from lympho-myeloid clonal hematopoiesis. Leukemia, 31, 1286-1295.

Wang, K., Sanchez-Martin, M., Wang, X., Knapp, K.M., Koche, R., Vu, L., Nahas, M.K., He, J., Hadler, M., Stein, E.M., Tallman, M.S., Donahue, A.L., Frampton, G.M., Lipson, D., Roels, S., Stephens, P.J., Sanford, E.M., Brennan, T., Otto, G.A., Yelensky, R., Miller, V.A., Kharas, M.G., Levine, R.L., Ferrando, A., Armstrong, S.A. & Krivtsov, A.V. (2017) Patient-derived xenotransplants can recapitulate the genetic driver landscape of acute leukemias. Leukemia, 31, 151-158.

Welch, J.S., Ley, T.J., Link, D.C., Miller, C.A., Larson, D.E., Koboldt, D.C., Wartman, L.D., Lamprecht, T.L., Liu, F., Xia, J., Kandoth, C., Fulton, R.S., McLellan, M.D., Dooling, D.J., Wallis, J.W., Chen, K., Harris, C.C., Schmidt, H.K., Kalicki-Veizer, J.M., Lu, C., Zhang, Q., Lin, L., O'Laughlin, M.D., McMichael, J.F., Delehaunty, K.D., Fulton, L.A., Magrini, V.J., McGrath, S.D., Demeter, R.T., Vickery, T.L., Hundal, J., Cook, L.L., Swift, G.W., Reed, J.P., Alldredge, P.A., Wylie, T.N., Walker, J.R., Watson, M.A., Heath, S.E., Shannon, W.D., Varghese, N., Nagarajan, R., Payton, J.E., Baty, J.D., Kulkarni, S., Klco, J.M., Tomasson, M.H., Westervelt, P., Walter, M.J., Graubert, T.A., DiPersio, J.F., Ding, L., Mardis, E.R. & Wilson, R.K. (2012) The origin and evolution of mutations in acute myeloid leukemia. Cell, 150, 264-278.

Wong, T.N., Miller, C.A., Klco, J.M., Petti, A., Demeter, R., Helton, N.M., Li, T., Fulton, R.S., Heath, S.E., Mardis, E.R., Westervelt, P., DiPersio, J.F., Walter, M.J., Welch, J.S., Graubert, T.A., Wilson, R.K., Ley, T.J. & Link, D.C. (2016) Rapid expansion of preexisting nonleukemic hematopoietic clones frequently follows induction therapy for de novo AML. Blood, 127, 893-897.

Robust acute myeloid leukemia engraftment in humanized scaffolds using injectable biomaterials and intravenous xenotransplantation

Stromal cells engineered to express T cell factors induce robust CLL cell proliferation in vitro and in PDX co-transplantations allowing the identification of RAF inhibitors as anti-proliferative drugs