Gene expression changes associated with progression and response in chronic myeloid leukemia Jerald P. Radich*†‡, Hongyue Dai§, Mao Mao§, Vivian Oehler*, Jan Schelter§, Brian Druker†¶, Charles Sawyersʈ, Neil Shahʈ, Wendy Stock**, Cheryl L. Willman†,††, Stephen Friend§, and Peter S. Linsley§ *Divisions of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; §Rosetta Inpharmatics, Seattle, WA 98109; ¶Oregon Health & Science University, University Center, Portland, OR 97239; ʈUniversity of California, Los Angeles, CA 90095; **University of Chicago School of Medicine, Chicago, IL 60637; ††University of New Mexico Cancer Research and Treatment Center, Albuquerque, NM 87131; and †Southwest Oncology Group, Ann Arbor, MI 48106 Communicated by E. Donnall Thomas, Fred Hutchinson Cancer Research Center, Seattle, WA, December 13, 2005 (received for review June 24, 2005) Chronic myeloid leukemia (CML) is a hematopoietic stem cell Imatinib Mesylate, which suppresses the Ph to the point where it is disease with distinct biological and clinical features. The biologic undetectable by cytogenetic evaluation (a complete cytogenetic basis of the stereotypical progression from chronic phase through remission or CCR) in Ͼ70% of newly diagnosed patients with accelerated phase to blast crisis is poorly understood. We used DNA chronic-phase CML disease (4). The long-term durability duration microarrays to compare gene expression in 91 cases of CML in of such responses is unknown, as is potential for cure with imatinib. chronic (42 cases), accelerated (17 cases), and blast phases (32 Complete molecular response, defined by the absence of the cases). Three thousand genes were found to be significantly (P < Bcr-Abl fusion mRNA by RT-PCR, is unusual in patients with CCR 10؊10) associated with phase of disease. A comparison of the gene (5), and patients who achieve a molecular response, but have signatures of chronic, accelerated, and blast phases suggest that imatinib discontinued, show a relatively rapid reemergence of the progression of chronic phase CML to advanced phase (accel- disease (6). Resistance to imatinib occurs (especially in advanced erated and blast crisis) CML is a two-step rather than a three-step phase disease) often accompanied by point mutations in the process, with new gene expression changes occurring early in tyrosine kinase domain of the abl gene (7). The natural history of accelerated phase before the accumulation of increased numbers such relapses is unknown, although some appear to progress rapidly of leukemia blast cells. Especially noteworthy and potentially into advanced stage disease (8). significant in the progression program were the deregulation of The genetic events that cause the progression of chronic phase to the WNT͞␤-catenin pathway, the decreased expression of Jun B blast crisis CML are largely unknown. Numerous genetic abnor- and Fos, alternative kinase deregulation, such as Arg (Abl2), and an malities have been demonstrated, including the acquisition of increased expression of PRAME. Studies of CML patients who additional chromosomal abnormalities including such as a dupli- Ј relapsed after initially successful treatment with imatinib demon- cation of the Ph , isochromosome 17(p) resulting in the disruption ͞ strated a gene expression pattern closely related to advanced of TP53, and less commonly, the deletion of the p15 p16 tumor phase disease. These studies point to specific gene pathways that suppressor genes (especially in lymphoid blast crisis) and the might be exploited for both prognostic indicators as well as new RUNX1-EV11 fusion (9, 10). Genetic instability is apparent, as targets for therapy. evidenced by the in the additional chromosomal changes that occur with progression, but standard assays of instability, such as alter- genetics ͉ blast crisis ͉ PRAME ͉ Wnt ations in minisatellite repeats, are detected infrequently (11, 12). Unfortunately, clinical and molecular tests cannot predict where on the ‘‘clock’’ of disease progression an individual lies at the time hronic myeloid leukemia (CML) is a hematopoietic stem cell of the diagnosis, and this makes it impossible to adapt therapy to the Cdisease with distinct biological and clinical features. CML degree of risk that faces an individual CML patient. We also cannot usually presents in chronic phase, in which the clonal expansion of yet identify the subset of patients who are most likely to benefit from mature myeloid cells leads to an elevated white blood cell count. a specific treatment option. This study is aimed at determining Without curative intervention, chronic-phase CML will invariably changes in gene expression that occur in the evolution of the chronic transform through a phase of ‘‘acceleration,’’ often heralded by the phase to blast crisis, with the hope of identifying genes and pathways appearance of increased immature myeloid cells in the bone that may be useful as prognostic markers or targets for therapeutics. marrow and peripheral blood, as well as new cytogenetic changes in addition to the Philadelphia chromosome (Ph). Progression then Results proceeds to blast crisis, with immature blast cells overwhelming the Phase-Specific Gene Expression in CML. We first examined the genes production of normal hematopoetic elements. Blast crisis is highly associated with the phase of disease. We used as a reference a pool resistant to treatment, with death generally occurring from infec- of 200 chronic phase bone marrows, and studied 91 individual cases tion and bleeding complications secondary to the absence of of CML in chronic phase (n ϭ 42), accelerated phase by blast count normal granulocytes and platelets. The median time from diagnosis criteria (n ϭ 9) or by the occurrence of additional clonal cytogenetic of chronic phase CML to progression to accelerated phase is Ϸ3–4 changes (n ϭ 8), blast crisis (n ϭ 28), and four cases of blast crisis years, but the range of timing is quite broad, encompassing from 0.5 in remission after chemotherapy. An ANOVA analysis revealed to 15 years (1). Ϸ3,500 genes (from a total of Ϸ24,000 genes) differentially ex- There are several treatment options for CML. All treatments are pressed across the different phases of disease by using a minimum more successful when administered during the chronic phase disease than in accelerated or blast phase. The only known curative therapy for CML is stem cell transplantation, a complex and Conflict of interest statement: No conflicts declared. potentially toxic modality that carries a high potential for morbidity Freely available online through the PNAS open access option. and mortality (2). Nontransplant therapy includes IFN-␣, which Abbreviations: CML, chronic myeloid leukemia; CCR, complete cytogenetic remission. produces a major reduction in the proportion of Ph-positive cells Data deposition: The sequence reported in this paper has been deposited in the Gene and extends the natural history of the disease in Ϸ10–20% of Ontology (GO) database (accession no. GSE4170). patients cases, with some alive and in remission for Ͼ10 years (3). ‡To whom correspondence should be addressed. E-mail: [email protected]. IFN-␣ has largely been replaced by the tyrosine kinase inhibitor, © 2006 by The National Academy of Sciences of the USA 2794–2799 ͉ PNAS ͉ February 21, 2006 ͉ vol. 103 ͉ no. 8 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510423103 Downloaded by guest on September 30, 2021 Fig. 1. Genes associated with CML progression. Sam- ples from patients with of CML cases in chronic phase, accelerated by cytogenetic criteria only, accelerated phase, blast crisis, and blast crisis ‘‘in remission’’ were compared to a pool of chronic phase RNA (See Mate- rials and Methods for details). Approximately 3,500 genes were significantly associated with progressive disease at a significance level of P Ͻ 10Ϫ11. Each row represents one sample, and each column represents one gene. Red color indicates overexpression relative to the control pool, and green color indicates low expression. statistical significance cutoff of P Ͻ 10Ϫ11 (Fig. 1 and Table 4, which drial genes, RNA-binding genes, and protein biosynthesis genes, is published as supporting information on the PNAS web site). This reflecting the increased proliferation and metabolism of progres- set of genes identified in the progression from chronic to blast phase sive disease. Advanced-phase CML, compared to chronic phase, is referred to here as the ‘‘phase reporter’’ gene set. exhibited a decreased expression of genes involved in structural We examined the proposed biologic function of the genes integrity and adhesion, as well as decreases in expression of genes associated with CML phases by applying a biological annotation involved in inflammatory and immune response. In addition, program based on the gene ontology (GO) and KEGG annotations. several protooncogenes and tumor suppressor genes are differen- The major functional groups of genes in the phase reporter gene set tially expressed in advanced phase CML, including N- and H-ras, are shown in Table 5, which is published as supporting information FLT3, yes, AF1q, CBFB, WT1, ORALOV1, Bcl-2, and PTPN11. on the PNAS web site. The functional groups most highly correlated with disease phase (accelerated͞blast phase relative to chronic CML Appears to Be a ‘‘Two-Step’’ Disease. We examined whether the phase) included increased expression of nuclear genes, mitochon- progression of CML best fit a three-step model as often described MEDICAL SCIENCES Fig. 2. Comparison of CML blasts to normal CD34ϩ cells and correction for the pattern of gene expression. (A) Phase genes were corrected for normal CD34ϩ gene expression (ANOVA, P Ͻ 1 ϫ 10Ϫ8). The gene expression of normal CD34ϩ cells was subtracted from each disease sample. The resulting pattern reflects genes associated with progression independent of normal blast biology. (B) Relative gene expression of the ‘‘top ten’’ genes found to be up-regulated in advanced phase disease compared to chronic phase. Radich et al. PNAS ͉ February 21, 2006 ͉ vol. 103 ͉ no. 8 ͉ 2795 Downloaded by guest on September 30, 2021 in the literature (chronic phase 3 accelerated phase 3 blast crisis), Table 1.