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Jun 19, 2008 - cytarabine and an anthracycline. No clinical trial is studying the combination of GO with cytarabine and L-asparaginase. The interpatient ...
Letters to the Editor

As we observed marked cross-resistance between calicheamicin and anthracyclines, combining these compounds may be less likely to result in increased clinical efficacy. Based on our in vitro data, we suggest combining GO with cytarabine and/or L-asparaginase. Currently, most trials combine GO with cytarabine and an anthracycline. No clinical trial is studying the combination of GO with cytarabine and L-asparaginase. The interpatient differences in calicheamicin sensitivity are the largest differences in drug sensitivity we have ever observed in pediatric AML, suggesting that it is likely that primary calicheamicin resistance plays a role in the response to GO. This needs to be validated in future clinical trials in which in vitro and in vivo response to GO/calicheamicin can be compared. In conclusion, when analyzing resistance to GO, primary resistance to calicheamicin should be considered as an important mechanism.

Acknowledgements This work was partially funded by ZonMW AGIKO Grant 920-03374 (BFG). Calicheamicin was provided free of charge by Wyeth Pharmaceuticals. BFG performed the experiments, analyzed the data and wrote the paper; CMZ designed the research and wrote the paper; SJHV performed the experiments, analyzed the data and edited the paper; AHL performed the experiments and edited the paper; UC, KH, DR and BESG provided the leukemic samples and clinical data and edited the paper; JC analyzed the data and edited the paper; GJLK designed the research and edited the manuscript.

Conflict of interest The authors state no conflict of interest.

BF Goemans1, CM Zwaan2, SJH Vijverberg1, AH Loonen1, U Creutzig3, K Ha¨hlen2,4, D Reinhardt3, BES Gibson5, J Cloos1 and GJL Kaspers1 1 Department of Pediatric Oncology/Hematology, VU university medical center, Amsterdam, The Netherlands; 2 Department of Pediatric Oncology/Hematology, Erasmus mc, Sophia Children’s Hospital, Rotterdam, The Netherlands; 3 AML-BFM Study Group, University of Muenster, Muenster, Germany; 4 DCOG, the Hague, The Netherlands and 5 MRC Childhood Leukaemia Working Party, Glasgow, UK E-mail: [email protected]

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References 1 Walter RB, Gooley TA, van der Velden V, Loken MR, van Dongen JJ, Flowers DA et al. CD33 expression and P-glycoprotein-mediated drug efflux inversely correlate and predict clinical outcome in patients with acute myeloid leukemia treated with gemtuzumab ozogamicin monotherapy. Blood 2007; 109: 4168–4170. 2 Becton D, Dahl GV, Ravindranath Y, Chang MN, Behm FG, Raimondi SC et al. Randomized use of cyclosporin A (CsA) to modulate P-glycoprotein in children with AML in remission: Pediatric Oncology Group Study 9421. Blood 2006; 107: 1315–1324. 3 Goemans BF, Zwaan CM, Harlow A, Loonen AH, Gibson BE, Hahlen K et al. In vitro profiling of the sensitivity of pediatric leukemia cells to tipifarnib: identification of T-cell ALL and FAB M5 AML as the most sensitive subsets. Blood 2005; 106: 3532–3537. 4 Linenberger ML. CD33-directed therapy with gemtuzumab ozogamicin in acute myeloid leukemia: progress in understanding cytotoxicity and potential mechanisms of drug resistance. Leukemia 2005; 19: 176–182.

A western blot assay for detecting mutant nucleophosmin (NPM1) proteins in acute myeloid leukaemia

Leukemia (2008) 22, 2285–2288; doi:10.1038/leu.2008.149; published online 19 June 2008

Acute myeloid leukaemia (AML) carrying a mutated NPM1 gene and aberrant dislocation of nucleophosmin (NPM1) into the cytoplasm1 accounts for about one-third of adult AML and shows distinctive biological and clinical features.2–4 AML with normal karyotype and mutated NPM1, in the absence of FLT3ITD, exhibits a favourable prognosis.2 Moreover, NPM1 mutations may serve as a marker for monitoring minimal residual disease.2 Thus, the search for NPM1 mutations has now become part of the initial diagnostic work-up in patients with AML. Approximately 40 different molecular variants of NPM1 mutations,2 the most frequent genetic alteration in AML, have been identified so far (Supplementary Table 1). Mutation A is the most common type (75–80% cases)1 and mutations B and D account for about 10 and 5% of NPM1-mutated AML, respectively; other mutations are very rare.2 Although several sensitive molecular techniques detect NPM1 mutations in DNA or RNA,2 a demand has arisen for simpler, less expensive diagnostic assays, which are suitable for centres that are not

equipped for molecular screening. Availability of such tests would contribute to expand the use of a genetic-based WHO classification of AML. Immunohistochemical detection of cytoplasmic NPM is a very simple, rapid, cheap, sensitive, specific assay that can be used as a surrogate for NPM1 mutational analysis.5,6 It is applicable to paraffin sections from bone marrow biopsies5 or extramedullary tissues7 but cannot be used on cytological samples (smears or cytospins).1,5 This is a potential limitation as not all haematological centres perform bone marrow biopsy as a frontline diagnostic procedure in AML patients. The present study assessed the value of western blot in identifying NPM1 mutants in cytological AML samples, using rabbit polyclonal antibodies that react with mutated, but not wild-type, NPM1 proteins.8,9 We applied western blot analysis to leukaemic samples from 213 AML patients at first diagnosis (Table 1). A total of 135 consecutive patients were analyzed prospectively using freshly collected cells (see Supplementary Materials), whereas 78 patients were studied retrospectively using liquid nitrogen snap-frozen dry pellets of bone marrow or peripheral blood mononuclear cells that had been stored for the past 3 years. The Leukemia

Letters to the Editor

2286 Table 1

Western blot analysis of NPM1 mutants: results in 213 AML patients Patients (N)

Western blot

Expression of NPMa

AML with mutated NPM1 Prospectively analyzed Retrospectively analyzed

106 69 37

106/106 69/69 37/37

106/106 (n+c) 69/69 (n+c) 37/37 (n+c)

AML with germ line NPM1 Prospectively analyzed Retrospectively analyzed

107 66 41

0/0 0/0 0/0

107/107 (n) 66/66 (n) 41/41 (n)

AML type

Abbreviations: AML, acute myeloid leukaemia; NPM, nucleophosmin; (n+c), nuclear plus cytoplasmic; (n), nucleus-restricted. a Detected by immunohistochemistry.

inclusion criterion was the availability of good quality protein extracts, as defined by the absence of protein degradation when membranes were probed with an anti-NPM wild-type specific antibody (anti-NPM, Clone FC-61991; Invitrogen, Carlsbad, CA, USA). Western blot analysis was performed according to the standard procedure on whole-cell lysate from bone marrow or peripheral blood samples (Supplementary Materials), using two rabbit polyclonal antibodies (Sil-A and Sil-C)8,9 raised against synthetic peptides corresponding to the C-terminal portion of the NPM1 mutant A (Figure 1a and Supplementary Materials). The specific wild-type NPM monoclonal antibody was used as the positive control. Results of the western blot were compared blindly with the results of immunohistochemical analysis of NPM1 subcellular expression, which was available in all patients. The presence of mutated NPM1 was confirmed by molecular analysis in the 42 cases investigated. In 106/213 AML patients, western blot analysis identified a specific band of 37 kDa molecular weight corresponding to a mutated NPM1 protein (Figure 1b). Immunohistochemistry confirmed the results of western blot in all cases, showing aberrant cytoplasmic expression of NPM1, which is fully predictive of NPM1 mutations5,6 (Table 1). Although band intensity was strong in almost all cases bands were less intense in one patient with low bone marrow leukaemic infiltration (about 25%) and in some cases with FAB-M5b morphology. The latter finding is in keeping with our previous immunohistochemical observation that NPM1 is downregulated during maturation from the leukaemic monoblast to pro-monocytes.1 No band was detected in 107/213 AML samples. On immunohistochemical analysis, all these cases showed nucleusrestricted expression of NPM1, which is fully predictive of NPM1 gene in germ line configuration (Table 1). In all 213 cases, western blot analysis was performed on Ficoll-isolated mononuclear cells (Figure 1b). To determine whether the procedure could be simplified, we performed western blot analysis of lysates of cells recovered from 1–2 drops of bone marrow or peripheral blood upon red cell lysis, in 67/ 213 cases. In all 67 cases, western blot predicted the NPM1 gene mutational status (29 positive and 38 negative) with similar efficiency as in isolated cells (Figure 1c). Results of all the studies were available within 24 h. As NPM1 mutations generate similar new sequences at the NPM1 C-terminus (Supplementary Table 1), we determined whether, in western blot analysis, Sil-A and Sil-C, rabbit polyclonal antibodies raised against peptide sequences of the most common NPM1 mutant A (286-DLCLAVEEVSLRK), identified leukaemic mutants other than the type-A mutant. Indeed, a strong positive signal identified NPM1 mutation B (Figure 2a, pt. 163), which generates a mutant with a slightly different C-terminal protein sequence (286-DLCMAVEEVSLRK) (Table 2). Thus, as our western blot-based approach identified mutations A Leukemia

Figure 1 (a) Rabbit polyclonal antibodies used for identification of NPM1 mutant proteins. Two rabbit polyclonal antibodies were generated against either a synthetic 11-amino-acid peptide (NH2-CLAVEEVSLRK-COOH named Sil-C; Inbio Ltd, Tallin, Estonia) or an 18-mer peptide (NH2-QEAIQDLCLAVEEVSLRK-COOH named Sil-A; Primm SRL, Milan, Italy), corresponding to the C-terminal of the NPM1 mutant A protein (Supplementary Materials). (b, c) Western blot identification of NPM1 mutant protein in AML patients. (b) Either bone marrow or peripheral blood from AML patients was subjected to separation on Ficoll-Hypaque, and the recovered mononuclear cells, containing leukaemic cells, directly lysed in Laemmli sample buffer. A total of 1–2  106 cells equiv. was loaded and run on SDSpolyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) and probed with anti-NPM mutant Sil-A antibody. Representative results of western blot (WB) analysis are shown. A band corresponding to a protein of 37 kDa MW is evident only in the NPM cytoplasmic-positive (NPMc þ ) AML group of samples. A positive signal at WB with a mouse monoclonal antibody recognizing only the wild-type NPM (anti-NPMwt) indicates good quality protein extraction. OCI/AML3 whole-cell lysate was included as positive control. (c) Whole blood from either bone marrow (Pre-F, pts. 83, 121 and 142) or peripheral blood (Pre-F, pt. 152) of NPMc þ AML patients was subjected to red cell lysis. White blood cells were lysed in Laemmli sample buffer, and WB analysis was performed as above. Results obtained using whole blood were compared directly with analysis on mononuclear cells recovered upon Ficoll separation from the same AML patients (Post-F). WB with the anti-NPMwt specific antibody demonstrates a very good quality of sample protein extraction.

Letters to the Editor

2287 and B, it may be expected to identify rare mutants carrying identical protein sequences to mutant A (mutants D, Om, 4, 7, Gw and Hw) or to mutant B (mutant Jw) (Table 2, Supplementary Table 1). Indeed, mutation D was identified in one patient (Figure 2a, pt. 76; Table 2). Although these mutations already account for over 95% of NPM1 mutations, we investigated whether western blot identified the other NPM1 mutant sequences (Supplementary Table 1) and analyzed cellular lysates of NIH-3T3 or Phoenix cell lines that had been transiently transfected with NPM1 gene constructs encoding NPM1 mutants E (286-DLWQSLAQVSLRK), G (286-DLWQCFAQVSLRK), L (286-DLSRAVEEVSLRK) and P (286-DLCTFLEEVSLRK) as green fluorescent protein-fusion proteins (Figure 2b), using cells transfected with NPM1 mutant A as control. Sil-A and Sil-C antibodies identified all these mutants, although the western blot signal was less intense for mutations E and G (Figure 2b), possibly because the acquired C-terminal sequences were markedly different from NPM1 mutant A. As expected, western blot did not identify the very rare exon-11 NPM1 mutation10,11 (NPM_mutVi2; Figure 2b), as it generates a truncated mutant form of NPM1 lacking the epitope sequence recognized by Sil-A and Sil-C antibodies. Thus, with the exception of the NPM1 exon-11 mutant, western blot may be presumed to identify all NPM1 mutations. In conclusion, our study provides evidence that western blot with specific antibodies is a highly flexible, simple and rapid assay for identifying NPM1 mutants in AML samples except for

exon-11 NPM1 mutant, which appears to be of little clinical relevance as only two AML patients with this mutation have been reported worldwide.10,11 Although objections could be raised against the use of polyclonal antibodies for diagnostic purposes, the results of our western blot analysis were highly reproducible with batches of reagents derived from different rabbits that had been immunized with the peptide corresponding to mutation A. Generation of monoclonal antibodies against NPM1 mutants may further facilitate large-scale production and use. We expect that adding western blotting to the armamentarium of current procedures for detecting NPM1 mutations (mutational analysis and immunohistochemistry) will encourage widespread use of a genetic-based WHO classification of AML, which is an important step, as the detection of NPM1 and FLT3 mutation status is becoming more and more crucial in therapeutic decisions.12 These techniques should be considered complementary rather than competitive as they offer a flexible approach to diagnosis and provide each centre with the option of whatever single or combined approach most suitable. Using one of the available molecular techniques13 to search for NPM1 mutations in all AML patients at first presentation is probably the most informative approach and is recommended, when possible. Disadvantages include sequencing of all AML cases, costs and need for sophisticated equipment and expert laboratory staff. In haematological centres that are not equipped for advanced molecular analyses, western blot and/or immunohistochemistry could, because of their simplicity and low costs, serve as front-line large-scale screening assays to identify AML patients carrying NPM1 mutations. Choice of either technique will depend on available material, considering that immunohistochemistry can be performed only on paraffin-embedded sections,1 whereas western blot is more suitable for analysis of cytological samples. Cases that result positive on western blot analysis, or express aberrant cytoplasmic NPM at immunohistochemistry, could subsequently be referred to more specialized laboratories to identify the type of NPM1 mutation in order to Table 2

Figure 2 Ability of the rabbit polyclonal anti-NPM mutant antibody to recognize NPM1 mutants other than the type-A mutant. (a) Protein extracts from patients bearing NPM1 mutation D (pt. 76) and B (pt. 163) were analyzed by western blot as in Figure 1. (b) Western blot analysis on whole-cell lysates from Phoenix cells transfected with either GFP_NPM1 mutant A (NPM_mutA) or GFP_NPM1 mutants E (NPM_mutE), G (NPM_mutG), L (NPM_mutL), P (NPM_mutP) and Vi2 (NPM_mutVi2). A positive signal is detectable in all NPM1 exon-12 mutation cases as a band at about 64 kDa MW (GFP_NPM1 fusion proteins; arrow, lane 1–5, upper panel). The truncated form of the NPM mutant protein product of NPM1 exon-11 mutation was not recognized (arrow, lane 6, upper panel). Western blotting with anti-GFP antibody documented good transfection efficiency and equal loading (arrow, lower panel).

NPM1 mutant sequences identified by WB analysis

NPM1 mutationa

C-terminus sequence

A D Om 4 7 Gw Hw

Number

WB

286-DLCLAVEEVSLRK 286-DLCLAVEEVSLRK 286-DLCLAVEEVSLRK 286-DLCLAVEEVSLRK 286-DLCLAVEEVSLRK 286-DLCLAVEEVSLRK 286-DLCLAVEEVSLRK

38 2 NA NA NA NA NA

++ ++ ++b ++b ++b ++b ++b

B Jw

286-DLCMAVEEVSLRK 286-DLCMAVEEVSLRK

1 NA

++ ++c

L P E G

286-DLSRAVEEVSLRK 286-DLCTFLEEVSLRK 286-DLWQSLAQVSLRK 286-DLWQCFAQVSLRK

NA NA NA NA

++d ++d +d +d

Re

286-DLCSAVEEVSLRK

1

++

Abbreviation: WB, western blot. NA ¼ not available in our series of patients; ++stronger signal; +weaker signal. a See Supplementary Table 1. b Predicted strong positivity according to sequence identical to mutant A. c Predicted strong positivity according to sequence identical to mutant B. d Tested on transfected cells. e Newly identified NPM1 gene mutation (insertion of CTCG at position 956 through 959 of the reference sequence). Leukemia

Letters to the Editor

2288 design primers for monitoring minimal residual disease. Finally, anti-NPM mutant antibodies might be useful for research purposes, for example, to isolate NPM1 mutant-specific protein complexes for proteomic analysis.

Acknowledgements This work was supported by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.) and Fondazione Monte dei Paschi di Siena. We thank Roberta Pacini and Manola Carini for performing immunohistochemical staining. We also thank Dr Geraldine Boyd, for editing the manuscript, and Mrs Claudia Tibido`, for secretarial assistance. Brunangelo Falini applied for a patent on the clinical use of NPM1 mutants.

MP Martelli1, N Manes1,8, A Liso2,8, V Pettirossi1, B Verducci Galletti1, B Bigerna1, A Pucciarini1, MF De Marco1, MT Pallotta1, 1 3 N Bolli , M Sborgia , F di Raimondo4, F Fabbiano5, G Meloni6, G Specchia7, MF Martelli1 and B Falini1 1 Section of Haematology and Clinical Immunology, University of Perugia, IBiT Foundation, Fondazione IRCCS Biotecnologie nel trapianto, Perugia, Italy; 2 Institute of Haematology, University of Foggia, Foggia, Italy; 3 Haematology Branch, Ospedale di Pescara, Pescara, Italy; 4 Haematology Branch, Ospedale Ferrarotto S. Bambino, Catania, Italy; 5 Haematology Branch, Ospedale V. Cervello, Palermo, Italy; 6 Haematology, ‘La Sapienza’ University, Rome, Italy and 7 Institute of Haematology, University of Bari, Bari, Italy E-mail: [email protected] 8 These authors contributed equally to this work. References 1 Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med 2005; 352: 254–266. 2 Falini B, Nicoletti I, Martelli MF, Mecucci C. Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin (NPMc+ AML): biologic and clinical features. Blood 2007; 109: 874–885.

3 Alcalay M, Tiacci E, Bergomas R, Bigerna B, Venturini E, Minardi SP et al. Acute myeloid leukemia bearing cytoplasmic nucleophosmin (NPMc+ AML) shows a distinct gene expression profile characterized by up-regulation of genes involved in stem-cell maintenance. Blood 2005; 106: 899–902. 4 Garzon R, Garofalo M, Martelli MP, Briesewitz R, Wang L, Fernandez-Cymering C et al. Distinctive microRNA signature of acute myeloid leukemia bearing cytoplasmic mutated nucleophosmin. Proc Natl Acad Sci USA 2008; 105: 3945–3950. 5 Falini B, Martelli MP, Bolli N, Bonasso R, Ghia E, Pallotta MT et al. Immunohistochemistry predicts nucleophosmin (NPM) mutations in acute myeloid leukemia. Blood 2006; 108: 1999–2005. 6 Falini B, Bolli N, Shan J, Martelli MP, Liso A, Pucciarini A et al. Both carboxy-terminus NES motif and mutated tryptophan(s) are crucial for aberrant nuclear export of nucleophosmin leukemic mutants in NPMc+ AML. Blood 2006; 107: 4514–4523. 7 Falini B, Lenze D, Hasserjian R, Coupland S, Jaehne D, Soupir C et al. Cytoplasmic mutated nucleophosmin (NPM) defines the molecular status of a significant fraction of myeloid sarcomas. Leukemia 2007; 21: 1566–1570. 8 Quentmeier H, Martelli MP, Dirks WG, Bolli N, Liso A, Macleod RA et al. Cell line OCI/AML3 bears exon-12 NPM gene mutation-A and cytoplasmic expression of nucleophosmin. Leukemia 2005; 19: 1760–1767. 9 Pasqualucci L, Liso A, Martelli MP, Bolli N, Pacini R, Tabarrini A et al. Mutated nucleophosmin detects clonal multilineage involvement in acute myeloid leukemia: impact on WHO classification. Blood 2006; 108: 4146–4155. 10 Albiero E, Madeo D, Bolli N, Giaretta I, Bona ED, Martelli MF et al. Identification and functional characterization of a cytoplasmic nucleophosmin leukaemic mutant generated by a novel exon-11 NPM1 mutation. Leukemia 2007; 21: 1099–1103. 11 Pitiot AS, Santamaria I, Garcia-Suarez O, Centeno I, Astudillo A, Rayon C et al. A new type of NPM1 gene mutation in AML leading to a C-terminal truncated protein. Leukemia 2007; 21: 1564–1566. 12 Schlenk RF, Dohner K, Krauter J, Frohling S, Corbacioglu A, Bullinger L et al. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. N Engl J Med 2008; 358: 1909–1918. 13 Wertheim G, Bagg A. Nucleophosmin (NPM1) mutations in acute myeloid leukemia: an ongoing (cytoplasmic) tale of dueling mutations and duality of molecular genetic testing methodologies. J Mol Diagn 2008; 10: 198–202.

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

Highly sensitive and quantitative detection of BCR-ABL kinase domain mutations by ligation PCR

Leukemia (2008) 22, 2288–2291; doi:10.1038/leu.2008.180; published online 10 July 2008

Acquired resistance to imatinib in patients with chronic myeloid leukaemia (CML) or Philadelphia-positive (Ph þ ) acute lymphoblastic leukaemia frequently arises due to point mutations of the BCR-ABL kinase domain (KD)1,2 so that, as new inhibitors with differential activity against KD mutant BCR-ABL become available, early detection and precise quantification of key mutations are likely to make an increasingly important contribution to patient-specific treatment decisions. A number of approaches have been developed in recent years to detect these mutations based on the techniques of DNA sequencing, denaturing high-performance liquid chromatography (DHPLC), mutation-specific PCR and mass array genotyping. However, Leukemia

although some of these assays can detect mutants in wild-type allele frequencies as low as 0.1% and others are quantitative, the combination of sensitivity and quantification has remained elusive. Reliable quantification has been provided to date by pyrosequencing, which has a detection limit of around 10% mutants in wild-type allele, and by some PCR-based approaches.3 The potential importance of the quantitative detection of low-level mutations has become clear from the use of PCR-based approaches using mutation-specific primers, which have identified low-level KD mutations in the majority of Ph þ acute lymphoblastic leukaemia patients before therapy and revealed that almost all of those harbouring a low-level T315I or p-loop mutation at diagnosis relapsed with the same mutation, even after achieving a molecular remission on imatinib and chemotherapy.2 Similar techniques have identified low-level, pretherapeutic KD mutations in CML, although the relevance to