Rearranging CBFA2T3 Is Specific for Acute Erythroid Leukemia

Translocation t(1;16)(p31;q24) rearranging CBFA2T3 is speciﬁc for acute erythroid leukemia

Leukemia (2011) 25, 1510–1512; doi:10.1038/leu.2011.100; domain, alpha subunit 2 translocated to 3 (CBFA2T3, previously published online 24 May 2011 known as MTG16, myeloid translocation gene). The breakpoint on chromosome 16 was identified inside the big intron 1 of the Acute erythroid leukemias are characterized by a predominantly CBFA2T3 gene. CBFA2T3 encodes a protein in the myeloid erythroid population of bone marrow cells. Most authors translocation gene family of transcriptional co-repressors. agree that two main subtypes exist: (1) erythroleukemia Recombination of CBFA2T3 with the RUNX1 gene has been (erythroid/myeloid) or acute myeloid leukemia (AML) M6a, demonstrated in six cases of AML with a t(16;21)(q24;q22)9 and according to the French-American-British criteria,1,2 in which then with the CBFA2T3 breakpoint either between exons 1 and 450% erythroid precursors in the entire nucleated cell 2 or between exons 3 and 4. The fusion retains four evolu- population and 420% myeloblasts in the non-erythroid cell tionarily conserved motifs, that is, NHR1, NHR2, NHR3 and the population are found, and (2) pure erythroid leukemia or AML zinc finger domain NHF4.9 In our case, the breakpoint was in M6b, according to the French-American-British classification,1,2 intron 1, thus making the product of the rearrangement similar to in which erythroblasts constitute more than 80% of the bone that previously described. marrow cells with no significant myeloblastic component.1–3 To find out which gene was involved from chromosome 1, we Whereas erythroleukemia (erythroid/myeloid) is predominantly selected three fosmid clones inside the chromosomal region a disease of adults and comprises 5–6% of all cases of AML,4 chr1:61 917 304–62 051 594 (1p31.3), as this was where the pure erythroid leukemia is extremely rare, is usually associated breakpoint position had been found using bacteria artificial with a rapid clinical course and can occur in all age groups.5 chromosomes clones. Two genes are present here, TM2D1 (beta- The Mitelman database of chromosome aberrations and amyloid binding protein precursor) and INADL (InaD-like). gene fusions in cancer provides karyotypic information on Fluorescence in situ hybridization experiments using the about 600 cases of acute erythroid leukemia.6 The most clone G248P88729H1 showed a specific hybridization signal common aberrations are structural and numerical changes on der(1)t(1;1), whereas clones G248P86539D6 and of chromosomes 5 and 7, followed by trisomy 8. G248P8780H2 gave no signal in the abnormal metaphases, but These aberrations are common also in other AML subtypes,7 a specific signal on 1p in the normal metaphases of the case; we however, and indeed it seems that no specific chromosomal therefore assume that the genomic regions corresponding to these aberration or pathogenetic mechanism (for example, fusion fosmids were deleted in the unbalanced 1;16-rearrangement. gene or gene deregulation) leading to erythroid leukemia has The two genes of primary interest as they are localized closest to been reported. the breakpoint, TM2D1 and INADL, were found deleted partially A 15-month-old boy was admitted to the local hospital with a or completely. The resolution level of fosmid clones does not history of failure to thrive, prolonged diarrhea, vomiting and allow us to determine the position of the breakpoint with high fever. A diagnosis of pure erythroid leukemia (AML M6b; precision, but the INADL gene had lost its 30 end, whereas the 50 Figure 1) was made and treatment was begun according to the end was still present on the der(1)t(1;1). A large part of TM2D1 NOPHO-AML 2004 protocol (Nordic Society of Pediatric was also lost through the rearrangements, but it is possible that Hematology and Oncology),8 but the patient died of resistant sequences near the 50 and 30 end of this gene are still present on disease after 5 months. der(1)t(1;1) and/or t(1;16). INADL encodes a protein with multiple The G-banding analysis of short-term cultured bone marrow PDZ domains, which mediate protein–protein interactions, and cells revealed an abnormal karyotype interpreted as 46,XY,- proteins with multiple PDZ domains often organize multimeric der(1)t(1;1)(p31;q21),t(1;16)(p31;q24)[11]/46,XY[9]. Fluorescence complexes at the plasma membrane. The INADL protein in situ hybridization, using the CBFB Dual Color Break Apart localizes to tight junctions and to the apical membrane Rearrangement probe, indicated no rearrangement of CBFB; of epithelial cells.10 The TM2D1 gene encodes a b-amyloid hence, the found translocation could not be a variant of the peptide-binding protein.11 It contains a structural module related known AML-associated inv(16) or t(16;16). A series of fluores- to that of the seven transmembrane domain G protein-coupled cence in situ hybridization-experiments with probes derived receptor superfamily known to be important in heterotrimeric from bacteria artificial chromosomes was performed to identify G protein activation (UCSC Genome Browser at http://genome. the exact breakpoints of the t(1;16)(p31;q24); these were found ucsc.edu/cgi-bin/). with clones RP11-430G17 (mapping to 1p31.3) and RP11- Unfortunately, RNA obtained from leukemic blood was 830F9 (mapping to 16q24.3; Figure 2). The fluorescence in situ heavily degraded and could not be used for reverse transcription hybridization findings led us to reinterpret the 1;16-rearrange- PCR or rapid amplification of cDNA ends studies, something ment as an unbalanced translocation and the karyotype was that would have been necessary to determine the molecular revised as 46,XY,der(1)t(1;1)(p31;q21),del(1)(p11p31),der(16)t(1;16) consequences of the karyotypic alterations. However, two main (p31;q24) (Figure 3). putative leukemogenic mechanisms can be envisaged in view of To identify the exact breakpoint on the der(16q), appropriate the findings described above. The first is that a fusion gene with fosmid clones were selected inside the region chr16: CBF2T3 as its 30 moiety might have been generated by the 87 493 639–87 591 195 (16q24), as this was where the break- 1;16-translocation. The fact that CBFA2T3 is known to operate point lay based on bacteria artificial chromosomes data. Only via a fusion gene mechanism in other types of AML9 and then one gene is present in this region, the core-binding factor, runt- showing a similar breakpoint position (between exons 1 and 2) Letters to the Editor 1511 to that of our case, adds to the attraction of this hypothesis. The coiled-coil motif in the N-terminal region and ankyrin-repeats two candidate partners of CBFA2T3 in such fusion would be in the C-terminal region, with an additional motif, KN, at the INADL and TM2D1. None of them is known to participate in N-terminus.13 Whereas the human Kank1 gene (KANK1) leukemogenesis in any other gene fusion or, for that matter, in was found as a candidate tumor suppressor gene for renal any other way. The second mechanism that our findings cell carcinoma mapping to chromosome band 9p24, the other indicate could be operative in the present case, is loss of members were discovered on the basis of domain and tumor suppressor activity from the region of 1p shown to be phylogenetic analyses. Deletions of the KANK1 locus have homozygously deleted. This region contains four genes: been reported in renal cell carcinoma, cervical carcinoma, TM2D1, INADL, L1TD1 (Line-1 type transposase domain bladder cancer, hepatocellular carcinoma, pancreatic carcinoma, containing 1) and KANK4 (ankyrin repeat domain 38). Although lung cancer, acute lymphocytic leukemia and breast cancer. the first two genes may be partially deleted and, therefore, also We found another member of the Kank family, KANK4, potentially could be fused with CBFA2T3 on 16q, the last two homozygously deleted in our case. Whether this deletion was are lost completely. L1TD1 belongs to the transposase 22 family pathogenetically important, remains unknown. and the only information about it presently available is that Regardless of which genes are pathogenetically involved in LINE-1 hypomethylation is associated with increased CpG the t(1;16)(p31;q24), and also regardless of whether the island methylation in Helicobacter pylori-related enlarged-fold important outcome is the generation of a leukemogenic fusion gastritis.12 KANK4 is one of the genes encoding the Kank family gene (the hypothesis we favor) or something else, it seems of proteins which are characterized by their unique structure, beyond any reasonable doubt that the 1;16-translocation is specific for acute erythroid leukemia. A search of the Mitelman database shows that a 1;16-rearrangement was found in five cases,6 and in two of these a t(1;16)(p31;q24) was reported, that is, the very same translocation found in our patient. Ko¨ller

Figure 1 (a) Bone marrow smear (600 Â , Wright–Giemsa stain). The smear shows abnormal proerythroblasts. The erythroblasts are of medium to large size, display dispersed chromatin, prominent nucleoli Figure 3 Partial karyotype of the der(1)t(1;1)(p31;q21), del(1) and cytoplasmic vacuoles. (b) Bone marrow section (400 Â , HE stain). (p11p31) and der(16)t(1;16) found in the described case. G-banded A predominant population of immature erythroblasts with round to chromosomes are presented on the left, whereas ideograms of the irregular nuclei and basophilic cytoplasm replaces the normal rearranged chromosomes are shown to the right. Arrows point at hematopoiesis. breakpoint positions.

Figure 2 Fluorescence in situ hybridization images of metaphase plates from the AML M6. (a) This hybridization was performed using locus- specific probes derived from bacteria artificial chromosomes clones CTD-3010L24 (labeled in green), RP11-830F9 (labeled in blue), CTD-2555A7 (labeled in red) and RP11-1122C1 (labeled in yellow), all mapping to 16q24. Although the clones labeled in blue and green are hybridizing to the normal 16 and the der(16)t(1;16), the clones labeled in red and yellow hybridize only to the normal 16, indicating that the clones are missing from the rearranged chromosome, that is, the deletion starts (the breakpoint is located) between clones RP11-830F9 (labeled in blue) and CTD-2555A7 (labeled in red). (b) This hybridization was performed using bacteria artificial chromosomes clones RP11-430G17 (labeled in red), RP11-446E24 (labeled in green) and CTD-3251C12 (labeled in yellow) mapping to 1p32.3-1p33 and hybridizing only to the der(16)t(1;16); the clones proximal to RP11-430G17 were deleted, that is, the breakpoint on chromosome 1 is near or inside clone RP11-430G17. (c) This hybridization was performed using bacteria artificial chromosomes clones RP11-230B22 (labeled in yellow), RP11-434H24 (labeled in green) and RP11-746E15 (labeled in red), all mapping to 1p31.3 and hybridizing only to the der(1)t(1;1), that is, the last clones present in the rearrangement before the homozygous deletion.

Leukemia Letters to the Editor 1512 et al.14 reported a 10-year-old child with acute erythroid 2 Cheson BD, Cassileth PA, Head DR, Schiffer CA, Bennett JM, leukemia and the karyotype 47,XY,t(1;16)(p31;q2?2),del(7) Bloomfield CD et al. Report of the National Cancer Institute- (q31), þ 19,del(20)(p11). Castaneda et al.15 reported a 6-year- sponsored workshop on definitions of diagnosis and response in old child with an acute undifferentiated leukemia, but where the acute myeloid leukemia. J Clin Oncol 1990; 8: 813–819. 3 Mazzella FM, Kowal-Vern A, Shrit MA, Wibowo AL, Rector JT, leukemic cells demonstrated an early erythroid lineage differ- Cotelingam JD et al. Acute erythroleukemia: evaluation of 48 cases entiation. The abnormal karyotype found at diagnosis and with reference to classification, cell proliferation, cytogenetics, relapse was 51,XY,t(1;16)(p31;q24), þ 6, þ 10, þ 15, þ 19, þ 21. and prognosis. Am J Clin Pathol 1998; 110: 590–598. The present case is therefore the third of only three with a 4 Stanley M, McKenna RW, Ellinger G, Brunning RD. Classification karyotype containing a t(1;16)(p31;q24), and all of them seem of 358 Cases of Acute Myeloid Leukemia by FAB criteria: to have been the highly uncommon AML M6. Whether the Analysis of Clinical and Morphological Findings in Chronic and Acute Leukemias in Adults. Martin Nijhiff Publishers: Boston, association is even more distinct to pure erythroid leukemia 1985. (AML M6b) as in our patient, or only to acute erythroid 5 WHO classification. Tumours of Haematopoietic and Lymphoid leukemias in general, cannot be assessed on the basis of the low Tissues. Lion: IARC Press, 2001. number of patients hitherto described. 6 Mitelman F, Johansson B, Mertens F. Mitelman database of chromosome aberrations in cancer. http://cgap.nci.nih.gov/ Chromosomes/Mitelman. Conflict of interest 7 Johansson B, Harrison CJ. Acute Myeloid Leukemia. In: Heim S, Mitelman F (eds). Cancer Cytogenetics - Chromosomal and Molecular Genetic Aberrations of Tumor Cells, 3rd edn. Wiley- The authors declare no conflict of interest. Blackwell; New Jersey, 2009, pp 45–139. 8 Barnard DR, Alonzo TA, Gerbing RB, Lange B, Woods WG. Comparison of childhood myelodysplastic syndrome, AML FAB Acknowledgements M6 or M7, CCG 2891: report from the Children’s Oncology Group. Pediatr Blood Cancer 2007; 49: 17–22. This work was supported by grants from the Norwegian Cancer 9 Gamou T, Kitamura E, Hosoda F, Shimizu K, Shinohara K, Society and the South-East Norway Regional Health Authority. Hayashi Y et al. The partner gene of AML1 in t(16;21) myeloid We thank Roberta Roberto, Tiziana Storlazzi, Hege Kilen malignancies is a novel member of the MTG8(ETO) family. Blood 1998; 91: 4028–4037. Andersen and Marthe Løvf for their technical support. 10 Chen Z, Leibiger I, Katz AI, Bertorello AM. Pals-associated tight 1,2 1,2 1,2 junction protein functionally links dopamine and angiotensin II F Micci , J Thorsen , L Haugom , to the regulation of sodium transport in renal epithelial cells. B Zeller3, A Tierens4 and S Heim1,2,5 1 Br J Pharmacol 2009; 158: 486–493. Section for Cancer Cytogenetics, Institute for Medical 11 Kajkowski EM, Lo CF, Ning X, Walker S, Sofia HJ, Wang W et al. Informatics, The Norwegian Radium Hospital, Beta -Amyloid peptide-induced apoptosis regulated by a novel Oslo University Hospital, Oslo, Norway; protein containing a g protein activation module. J Biol Chem 2 Centre for Cancer Biomedicine, 2001; 276: 18748–18756. University of Oslo, Oslo, Norway; 12 Yamamoto E, Toyota M, Suzuki H, Kondo Y, Sanomura T, 3Department of Pediatrics, Rikshospitalet, Murayama Y et al. LINE-1 hypomethylation is associated with Oslo University Hospital, Oslo, Norway; increased CpG island methylation in Helicobacter pylori-related 4Department of Pathology, The Norwegian Radium Hospital, enlarged-fold gastritis. Cancer Epidemiol Biomarkers Prev 2008; Oslo University Hospital, Norway and 17: 2555–2564. 5Faculty of Medicine, University of Oslo, Oslo, Norway 13 Kakinuma N, Zhu Y, Wang Y, Roy BC, Kiyama R. Kank proteins: E-mail: [email protected] structure, functions and diseases. Cell Mol Life Sci 2009; 66: 2651–2659. 14 Ko¨ller U, Haas OA, Ludwig WD, Bartram CR, Harbott J, References Panzer-Grumayer R et al. Phenotypic and genotypic heterogeneity in infant acute leukemia. II. Acute nonlymphoblastic leukemia. 1 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Leukemia 1989; 3: 708–714. Gralnick HR et al. Proposed revised criteria for the classification of 15 Castaneda VL, Parmley RT, Saldivar VA, Cheah MS. Childhood acute myeloid leukemia. A report of the French-American-British undifferentiated leukemia with early erythroid markers and c-myb Cooperative Group. Ann Intern Med 1985; 103: 620–625. duplication. Leukemia 1991; 5: 142–149.

ZRF4, a combi-molecule with increased efﬁcacy as compared with the individual components in chronic lymphocytic leukemia lymphocytes in vitro

Leukemia (2011) 25, 1512–1516; doi:10.1038/leu.2011.110; of chemotherapy with immunotherapy have shown higher published online 7 June 2011 response rates and longer duration of responses, they can be associated with poor tolerability characterized by deterioration B-cell chronic lymphocytic leukemia (CLL) is characterized by of immune functions leading to infections.2 More recently, actively dividing B-lymphocytes in the lymph nodes and bone bendamustine, a hybrid agent with a nitrogen mustard moiety marrow, as well as the accumulation of quiescent lymphocytes and a purine analog, has been tested in clinical trials in CLL, in the peripheral blood of affected patients. During treatment, demonstrating that hybrid molecules can result in increased the enzyme-mediated repair of DNA damage can induce therapeutic efﬁcacy as compared with the nitrogen mustard resistance to chemotherapeutic drugs.1 Even if combinations analog alone.3 We have previously demonstrated that imatinib,

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