Letters to the editor 489 NPM1 mutations occur rarely or not at all in chronic myeloid leukaemia patients in chronic phase or blast crisis

Leukemia (2013) 27, 489–490; doi:10.1038/leu.2012.193 this assay, which was consistent with the results of previous studies.1,6 We then extended the screen to a cohort of CP-CML patients Progression of chronic phase-chronic myeloid leukaemia (CP-CML) (n ¼ 33) to determine their NPM1 mutation status, including 17 to accelerated phase and blast crisis (BC) is frequently associated poor-risk patients defined by low OCT-1 activity values (omedian, with the acquisition of additional clonal chromosomal aberrations, 7.2 ng/200 000 cells). This group of poor-risk patients are highly and frequently involves mutations in RUNX1, ASXL1, WT1, NRAS, associated with the development of kinase domain mutations 9 KRAS, TET2, CBL, CBLB, TP53, IDH1/2, IKZF1, GATA2 and PRDM16,1,2 and disease progression. Furthermore, patients with low OCT-1 among others. Mutations in nucleophosmin 1 (NPM1; localised on activity have a poorer rate of achievement of major and complete 5q32) are the second most common mutations in acute myeloid molecular response up to 5 years, and lower event-free, leukaemia (AML) after FLT3 mutations,3 and are associated with transformation-free and overall survival. Once again we detected distinctive transcriptional signatures and a reduced ability of no NPM1 mutations in any of the 33 CP-CML patients, which was 4,8 NPM1 to shuttle between the nucleolus, nucleus and cytoplasm.4 consistent with previous small-cohort CP-CML studies. Included NPM is also thought to have a tumour-suppressor function in in our CP-CML cohort was a single patient who progressed from myeloid haematopoiesis, where a reduction in wild-type NPM may CP- to BC-CML 4 months after starting imatinib therapy. Matched contribute to tumourigenesis;4 however, the exact role is still unclear. samples at diagnosis and BC progression were available and mut Most recently, Georgiou et al.5 reported a case of a CML patient tested with this patient who remained NPM1 negative at both who achieved complete cytogenetic and molecular response time points. on 400 mg imatinib daily, and subsequently developed AML Our study and those of others show that mutations in the NPM1 with normal karyotype and mutated NPM1/negative FLT3-ITD gene are rarely associated with CP- or BC-CML patients, including those (NPM1mut/FLT3-ITDneg). This patient achieved a complete molecular at risk of disease progression (low OCT-1 activity). The case report of response 12 months after the initiation of imatinib treatment; CML transformation to AML associated with NPM1 mutation described 5 however, after 43 months that patient was subsequently by Georgiou et al. represents a very rare case. Such transformation of þ diagnosed with AML M2 (FAB classification). AML karyotype CML to AML (distinct from Ph BC-CML) is extremely rare and only a 11–15 analysis was normal (46,XY[20]) with BCR-ABL1 transcripts handful of cases have been reported. As the NPM1 mutation undetectable by both real-time quantitative PCR and nested status was not obtained in these cases, it is not possible to PCR. Further PCR analysis identified NPM1mut type B without ascertain whether it is predictive of transformation to AML. FLT3-ITD. Interestingly, subsequent testing demonstrated that the Therefore, we conclude that NPM1 mutations do not have a role in NPM1 mutation was present at low levels 12 months prior to AML CML disease initiation or progression to BC. This suggests that the diagnosis, suggesting a possible role in the transformation of CML. events that cooperate with BCR-ABL1 in acute transformation are NPM1 mutations have also been reported in Philadelphia distinct from those that drive the leukaemogenic process in a chromosome (Ph þ ) AML,6 indicating an ability to cooperate large subset of AML cases associated with normal karyotype and a with BCR-ABL1 in leukaemic transformation. One case of NPM1mut high frequency of NPM1 mutations. myeloid BC-CML has also previously been reported in the literature.7 In this particular case, the NPM1 mutation did appear to function in the transformation of BC-CML, as the mutation was CONFLICT OF INTEREST found in conjunction with the BCR-ABL1 rearrangement in almost The authors declare no conflict of interest. all blastic leukaemic cells. However, other studies have failed to definitively show NPM1 mutations in CML (both CP-4,8 and BC- CML1,6) using various techniques, but CP-CML patients at risk of ACKNOWLEDGEMENTS disease progression have not been fully interrogated. To This research was supported by the Leukaemia Foundation of Australia. investigate whether NPM1 mutations have a role in BC-CML or early in the progression of CP-CML, and in particular whether such DB Watkins1,2,3, TP Hughes1,2,3, DL White1,2,3 and RJ D’Andrea1,2,3,4 mutations may be associated with poor-risk CP-CML, we deter- 1Haematology Department, SA Pathology (RAH Campus), mined NPM1 mutations status in a cohort of BC- and CP-CML 9 Adelaide, South Australia, Australia; patients identified by low OCT-1 activity. 2Centre for Cancer Biology, SA Pathology, Adelaide, Here we report the screening of our BC-CML patient cohort for South Australia, Australia; mutations in the NPM1 gene locus. Genomic DNA collected from 3Department of Medicine, University of Adelaide, Adelaide, 14 BC-CML patient’s presenting to our centre was screened using South Australia, Australia and a fluorescence-based PCR assay, capillary electrophoresis (ABI 4Department of Haematology and Oncology, Prism 3100 Genetic Analyser, Applied Biosystems, Carlsbad, CA, The Queen Elizabeth Hospital, Woodville, South Australia, Australia USA) and GeneScan analysis (Applied Biosystems), as previously E-mail: [email protected] described.10 Out of these 14 patients, five patients were diagnosed with BC, five progressed from CP-CML and four were referred from other centres with BC. A single set of primers was REFERENCES used to screen for the most common NPM1 mutations (a four- 1 Grossmann V, Kohlmann A, Zenger M, Schindela S, Eder C, Weissmann S et al. nucleotide insertion located in nt959 or 965 of exon 12, covering 4 A deep-sequencing study of chronic myeloid leukemia patients in blast crisis all known NPM1 mutations), producing a 232-bp product for (BC-CML) detects mutations in 76.9% of cases. Leukemia 2011; 25: 557–560. mut wild-type NPM1 or 236-bp product for NPM1 . We observed no 2 Skorski T. Genetic mechanisms of chronic myeloid leukemia blastic transforma- NPM1 mutations in the 14 BC-CML patients analysed using tion. Curr Hematol Malig Rep 2012; 7: 87–93.

Accepted article preview online 13 July 2012; advance online publication, 31 July 2012

& 2013 Macmillan Publishers Limited Leukemia (2013) 482 – 516 Letters to the editor 490 3 Patel JP, Gonen M, Figueroa ME, Fernandez H, Sun Z, Racevskis J et al. Prognostic phase chronic myeloid leukemia treated with imatinib. J Clin Oncol 2010; 28: relevance of integrated genetic profiling in acute myeloid leukemia. N Engl J Med 2761–2767. 2012; 366: 1079–1089. 10 Lin LI, Lin TC, Chou WC, Tang JL, Lin DT, Tien HF. A novel fluorescence-based 4 Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L et al. Cytoplasmic multiplex PCR assay for rapid simultaneous detection of CEBPA mutations and nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J NPM mutations in patients with acute myeloid leukemias. Leukemia 2006; 20: Med 2005; 352: 254–266. 1899–1903. 5 Georgiou G, Efthymiou A, Vardounioti I, Boutsikas G, Angelopoulou MK, Vassila- 11 Dvorak P, Hruba M, Subrt I. Development of acute myeloid leukemia associated kopoulos TP et al. Development of acute myeloid leukemia with NPM1 mutation, with Ph-negative clone with inv(3)(q21q26) during imatinib therapy for chronic in Ph-negative clone, during treatment of CML with imatinib. Leukemia 2012; 26: myeloid leukemia. Leuk Res 2009; 33: 860–861. 824–826. 12 Fava C, Cortes J. Philadelphia-negative acute myeloid leukemia with new 6 Konoplev S, Yin CC, Kornblau SM, Kantarjian HM, Konopleva M, Andreeff M et al. chromosomal abnormalities developing after first-line imatinib treatment for Molecular characterization of de novo Ph þ acute myeloid leukemia. chronic phase chronic myeloid leukemia. Am J Hematol 2008; 83: 755. Leuk Lymphoma. e-pub ahead of print 9 July 2012; doi:10.3109/ 13 Kovitz C, Kantarjian H, Garcia-Manero G, Abruzzo LV, Cortes J. Myelodysplastic 10428194.2012.701739. syndromes and acute leukemia developing after imatinib mesylate therapy for 7 Piccaluga PP, Sabattini E, Bacci F, Agostinelli C, Righi S, Salmi F et al. Cytoplasmic chronic myeloid leukemia. Blood 2006; 108: 2811–2813. mutated nucleophosmin (NPM1) in blast crisis of chronic myeloid leukaemia. 14 Pawarode A, Sait SN, Nganga A, Coignet LJ, Barcos M, Baer MR. Acute myeloid Leukemia 2009; 23: 1370–1371. leukemia developing during imatinib mesylate therapy for chronic myeloid leuke- 8 Oki Y, Jelinek J, Beran M, Verstovsek S, Kantarjian HM, Issa JP. Mutations mia in the absence of new cytogenetic abnormalities. Leuk Res 2007; 31: 1589–1592. and promoter methylation status of NPM1 in myeloproliferative disorders. 15 Schafhausen P, Dierlamm J, Bokemeyer C, Bruemmendorf TH, Bacher U, Zander Haematologica 2006; 91: 1147–1148. AR et al. Development of AML with t(8;21)(q22;q22) and RUNX1-RUNX1T1 fusion 9 White DL, Dang P, Engler J, Frede A, Zrim S, Osborn M et al. Functional activity of following Philadelphia-negative clonal evolution during treatment of CML with the OCT-1 protein is predictive of long-term outcome in patients with chronic- Imatinib. Cancer Genet Cytogenet 2009; 189: 63–67.

Telomeres and chromosomal instability in chronic lymphocytic leukemia

Leukemia (2013) 27, 490–493; doi:10.1038/leu.2012.194 data identified groups with distinct cytogenetic and telomere profiles. This prospective study included 77 patients (57 males and 20 females, median age of 67 years) diagnosed with CLL at Telomeres are protective chromosomal end structures composed the Clermont-Ferrand University Hospital. Blood samples were of G-rich nucleotide repeats and an associated protein complex obtained after informed consent. At the time of sampling, 34 termed shelterin. Because of incomplete replication, telomeric patients were in Binet stage A, 17 in stage B and 26 in stage C. repeats are lost with every cell division, and this telomere attrition Samples were obtained before any treatment in 60 (77.9%) is involved in cell senescence and cancer (reviewed in Artandi and patients. Among the remaining 17 cases, 16 received no treatment DePinho1). Short, unprotected telomeres are erroneously fused within 6 months prior to the date of sampling. by the DNA-repair system, which gives rise to aneuploidies or Karyotype was analyzed after immunostimulation of cell structural chromosome rearrangements through chromosome cultures with the CpG-oligonucleotide DSP30 and interleukin 2. fusion-bridge-breakage cycles. Thus, telomere dysfunction can Interphase fluorescence in situ hybridization (FISH) was performed drive genomic instability during tumorigenesis.1 with a panel of commercially available probes (Abbott Molecular, In chronic lymphocytic leukemia (CLL), short telomeres have Rungis, France) for the detection of trisomy 12 and deletions of been associated with poor-prognosis cytogenetics.2,3 Besides 13q14, 11q22.3 (ATM) and 17p13 (p53). telomere length, the functional state of telomeres depends on Telomere studies were performed on mononuclear cells (MNCs). several molecular factors. Telomeres can be regenerated by Average telomere length was evaluated with quantitative real- telomerase and are protected from fusions by shelterin proteins. time DNA PCR.2 The method measures the difference between a Detailed investigations assessing all together the expression of sample and a reference normal DNA in the ratio of telomere telomerase and shelterin genes, telomere length, recurrent aberra- repeat copy number to single-gene copy number (relative T/S tions and overall karyotype instability have not yet been reported ratio). This T/S ratio is proportional to the average telomere length in CLL. Here, we studied the telomere status and chromosomal assessed with a classical telomere restriction fragment analysis aberrations in a series of CLL patients. A clustering analysis of the (r ¼ 0.89; Supplementary Figure S1).

Figure 1. (a–c) Hierarchical clustering of 77 CLL patients according to the pattern of chromosomal aberrations and telomere characteristics. Clustering was performed on the combined telomeres and cytogenetics data set (a) and separately: cytogenetics alone (b) and telomeres alone (c). Qualitative and quantitative values were normalized (centered and reduced) before clusterization. Aneuploidies, translocations or deletions, del17p or del11q, and trisomy 12 are presented in green (absence of abnormality) and red (presence of abnormality) squares. Presence of the low-risk del13q is shown in green, the absence in dark red. The different color intensities of dichotomic parameters reflect normalized values and not original binary (0/1) values. High normalized quantitative values of telomere length and expression of telomere- related genes are shown in red, intermediate values in black and lower values in green (intensity scale is shown). These normalized values, which are not logarithmic, are necessary to calculate distances between subjects and items. Distances between clusters were calculated using 1-Pearson’s correlation coefficient values and the dendrogram was constructed according to Ward’s algorithm. Of note, the average proportion of tumor cells in patient samples belonging to different clusters was essentially the same. For instance, the mean tumor cell content was 75% in cluster I, 75% in cluster II and 72% in cluster III (Kruskal–Wallis H-test, P ¼ 0.68, not significant). (d) The proportions of cases with negative prognostic factors in different clusters. P-values were obtained with the Chi-square test showing the overall difference between the clusters.

Accepted article preview online 13 July 2012; advance online publication, 10 August 2012

Leukemia (2013) 482 – 516 & 2013 Macmillan Publishers Limited