Correspondence V., Fonseca, R., Greipp, P.R., Witzig, T.E., Lust, J.A., Zeldenrust, S.R., Snow, D.S., Hayman, S.R., McGregor, C.G. & Jaffe, A.S. (2004a) Prognostication of survival using cardiac troponins and N-terminal pro-brain natriuretic peptide in patients with primary systemic amyloidosis undergoing peripheral blood stem cell transplantation. Blood, 104, 1881–1887. Dispenzieri, A., Gertz, M.A., Kyle, R.A., Lacy, M. Q., Burritt, M.F., Therneau, T.M., Greipp, P.R., Witzig, T.E., Lust, J.A., Rajkumar, S.V., Fonseca, R., Zeldenrust, S.R., McGregor, C.G. & Jaffe, A. S. (2004b) Serum cardiac troponins and N-terminal pro-brain natriuretic peptide: a staging system for primary systemic amyloidosis. Journal of Clinical Oncology, 22, 3751–3757. Gertz, M., Lacy, M., Dispenzieri, A., Hayman, S., Kumar, S., Buadi, F., Leung, N. & Litzow, M.
(2008) Troponin T level as an exclusion criterion for stem cell transplantation in light-chain amyloidosis. Leukaemia & Lymphoma, 49, 36– 41. Jimenez-Zepeda, V.H., Franke, N., Delgado, D., Winter, A., Stewart, K., Mikhael, J., Reece, D., Trudel, S., Chen, C. & Kukreti, V. (2010) HighDose Melphalan for AL Amyloidosis: the Importance of Case Selection to Improve Clinical Outcomes. Blood, 116, 996. Abstract 2403. Lachmann, H.J., Gallimore, R., Gillmore, J.D., Carr-Smith, H.D., Bradwell, A.R., Pepys, M.B. & Hawkins, P.N. (2003) Outcome in systemic AL amyloidosis in relation to changes in concentration of circulating free immunoglobulin light chains following chemotherapy. British Journal of Haematology, 122, 78–84.
Mollee, P., Marlton, P., Mills, A., Bird, R. & Gill, D. (2005) Unexpected hematologic toxicity associated with the use of intravenous intermediate dose melphalan and dexamethasone in patients with cardiac AL amyloidosis. Haematologica, 90, 202. Abstract PO.1409. Sattianayagam, P., Hawkins, P. & Gillmore, J. (2009) Amyloid and the GI tract. Expert Review of Gastroenterology & Hepatology, 3, 615–630. Schey, S.A., Kazmi, M., Ireland, R. & Lakhani, A. (1998) The use of intravenous intermediate dose melphalan and dexamethasone as induction treatment in the management of de novo multiple myeloma. European Journal of Haematology, 61, 306–310.
t(12;13)(q14;q31) leading to HMGA2 upregulation in acute myeloid leukaemia
The high mobility group A (HMGA) family of genes, including HMGA2 (formerly HMGI-C) on the long arm of chromosome 12, encodes four different chromatin binding proteins (Fusco & Fedele, 2007). HMGA2 is best known for its pathogenetic involvement in benign mesenchymal tumours, such as lipomas (Fusco & Fedele, 2007), with chromosomal rearrangements of 12q13-15 and has been extensively studied in that context. The Mitelman database (Mitelman et al, 2012) reports that 12q13-15 rearrangements are found also in 200 cases of acute myeloid leukaemia (AML). However, molecular investigations of haematological malignancies showing involvement of HMGA2 through various translocations are limited to 14 cases (Kottickal et al, 1998; Storlazzi et al, 2006; Aliano et al, 2007; Mitelman et al, 2012). We present a case of AML with a novel balanced t(12;13)(q14;q31) as the only cytogenetic aberration. The translocation gave rise to a truncated HMGA2 transcript. A 28-year-old man was diagnosed with AML-M1 after a blood sample showed anaemia (Hb 72 g/l), neutropenia (9·6 9 109/l), and thrombocytopenia (24 9 109/l) and a blood smear showed blasts among the normal cells. No lymphadenopathy, hepatomegaly or splenomegaly was found. Examination of a bone marrow aspirate revealed 90% immature myeloid blasts. A routine search for RUNX1-RUNX1T1, MLL-AFF1, MLL-MLLT3, PML-RARA, CBFB-MYH11, and BCR-ABL1 fusion genes and the NPM1 mutation was negative, but a tyrosine kinase domain mutation in FLT3 was identified. Induction therapy was initiated according to the Mayer protocol (Mayer et al, 1994). Remission was not ª 2012 Blackwell Publishing Ltd, British Journal of Haematology, 2012, 157, 762–774
obtained, so a new induction was tried. Complete remission was obtained two months after diagnosis. However, after the first round of maintenance therapy, the patient was readmitted with neutropenia and fever. He died shortly afterwards. The cytogenetic analysis of bone marrow cells revealed a t(12;13)(q14;q31) as the sole karyotypic abnormality, a translocation not previously reported in leukaemias (Mitelman et al, 2012). Fluorescence in situ hybridization analyses using bacterial artificial chromosomes (BACs) selected from the University of California, Santa Cruz database (www.genome. ucsc.edu) were used to map breakpoint positions. The breakpoint area on 12q was identified within BAC RP11-236B17, which contains the 3′ end of the HMGA2 gene (Fig. 1). To map the breakpoint more precisely, fosmid clones of about 40 kb were hybridized to metaphase plates; clone G248P8386A6 gave a split signal on chromosomes 12, der(12), and der(13) (Fig. 1). The area where the break had occurred was thus limited to about 40 kb overlapping the HMGA2 gene. On chromosome 13 BAC clone RP11-715M24 mapping to 13q31.1 gave a split signal. No genes are known in this area and we therefore hypothesized that HMGA2 upregulation, rather than formation of a fusion gene, was the pathogenetic mechanism in this case. To explore the rearrangements on the 3′ end of the HMGA2 transcript we performed a 3′ RACE (rapid amplification of cDNA ends) reaction using the SMARTer RACE cDNA Amplification kit (Clontech Laboratories, Mountain View, CA, USA). RNA was extracted from bone marrow obtained at diagnosis. Using 3′ RACE polymerase chain reaction (PCR), a band of approximately 1·2 kb was amplified 769
Correspondence
(A)
(B)
(C)
Fig 1. Molecular findings in the t(12;13) case. (A) Polymerase chain reaction (PCR) products of amplified sequences. Lane 1: Full-length HMGA2 transcript was detected in bone marrow from the patient using primers located in exons 1 and 5. Lane 2: In the 3′ RACE PCR a band of about 1·2 kb was amplified using a HMGA2-specific primer mapping to exon 2 in combination with the 3′RACE universal primer. The resulting product was extracted from the gel and sequencing confirmed an abnormal HMGA2 transcript. Lanes 3 and 4 show a positive and negative control, respectively. L: Ladder (B) Expression levels of HMGA2 in the t(12;13) case and acute myeloid leukaemia (AML) controls relative to normal bone marrow. (C) Schematic overview of the breakpoint region on chromosome 12. The upper panel shows the BAC and fosmid clone that gave split signals relative to the HMGA2 gene. The location of the transcript obtained by sequencing is illustrated in the lower panel relative to the HMGA2 gene and the wild-type transcript. The sequence is predicted to code for seven amino acids from intron 3 before meeting a stop codon.
with a HMGA2 gene-specific primer located within exon 2 and a universal primer mix (Fig. 1A). Sequencing analysis showed that the fragment contained exon 2, the full length of exon 3, and part of intron 3. The transcript is predicted to code for the full length of exons 1-3 in addition to seven amino acids of intronic sequence before meeting a stop codon (Fig. 1C), i.e., the truncated form of HMGA2 transcript is lacking its 3′ terminal acidic tail. Full length HMGA2 transcript was also amplified from patient cDNA (Fig. 1A). No expression was detected in normal bone marrow cDNA (Biochain, San Diego, CA, USA) used as control, which was expected because HMGA2 is not expressed in adult bone marrow (Fusco & Fedele, 2007). To quantify the expression level of HMGA2 we used the TaqMan system (HT7900; Applied Biosystems, Foster City, CA, USA) with primers assays spanning exons 1–2 (Hs00171569_m1) and 4–5 (Hs00971725_m1). Three patients 770
with a diagnosis of AML but without 12q rearrangements were used as controls. All samples were run in triplicate and the 2 DDct method was used as an estimate for difference in gene expression level. GUSB (Applied Biosystems, catalogue no: 4333767T) was used as a control for normalization and normal bone marrow RNA (Biochain) as calibrator. Both primer assays showed considerably increased expression levels compared to the AML controls (Fig. 1B). HMGA2 protein expression was evaluated by immunohistochemistry using a polyclonal rabbit HMGA2 antibody (catalogue no: 59170AP; Biocheck, Inc., Foster City, CA, USA) as described (Gorunova et al, 2011). A bone marrow biopsy was deparaffinized and specific staining was observed in the blast cell nuclei, confirming the expression of HMGA2 protein caused by the t(12;13)(q14;q22). The t(12;13)(q14;q22) detected in the presently described case of AML has not been reported earlier but led to a trunª 2012 Blackwell Publishing Ltd, British Journal of Haematology, 2012, 157, 762–774
Correspondence cated HMGA2 transcript. Only few cases of leukaemia have been molecularly investigated for involvement of HMGA2, but given that some 200 AML cases with rearrangement of 12q13-15 are reported in the literature (Mitelman et al, 2012) it is possible that a substantial subset of leukaemias exists in which upregulation of HMGA2 is the pathogenetic mechanism. Before the identification of the HMGA2 gene, Seyger et al (1995) reported findings in 25 AML patients with translocations involving 12q13. A majority displayed myeloblasts of minimal maturation (French-American-British type M1/ M2) and had short survival, features also seen in our patient. We found HMGA2 expression to be 10-fold higher than in other AML cases without 12q rearrangements. Truncated HMGA2 transcripts containing only exons 1-3 have been reported to have oncogenic properties (Mayr et al, 2007). In addition, HMGA2 transcripts lacking the native 3′ UTR could evade translational suppression by microRNA let7 (Mayr et al, 2007). High expression of exons 4 and 5 was also seen in our quantitative PCR study, suggesting an upregulation of wild-type HMGA2 through a currently unknown mechanism. No study has related HMGA2 expression to survival in leukaemias. An inverse relationship between HMGA2 expression and prognosis has among other malignancies been observed in patients with gastric cancer (Motoyama et al, 2008). Against this background it is possible that HMGA2 expression also has both prognostic and pathogenetic impact in leukaemia, but only systematic surveys of larger groups of patients can clarify this issue. As HMGA2 expression is normally absent or low in adult haematopoietic tissues (Fusco & Fedele, 2007), a drug targeting HMGA2 expression could be therapeutically useful.
Acknowledgements The authors thank the Norwegian Cancer Society and the Southeastern Norway Regional Health Authority for grants supporting the study, and Mette S. Førsund for technical assistance with the immunohistochemistry experiments.
References Aliano, S., Cirmena, G., Garuti, A., Fugazza, G., Bruzzone, R., Rocco, I., Malacarne, M., Ballestrero, A. & Sessarego, M. (2007) HMGA2 overexpression in polycythemia vera with t(12;21)(q14;q22). Cancer Genetics and Cytogenetics, 177, 115–119. Fusco, A. & Fedele, M. (2007) Roles of HMGA proteins in cancer. Nature Reviews Cancer, 7, 899–910. Gorunova, L., Bjerkehagen, B. & Heim, S. (2011) Paratesticular leiomyoma with a der(14)t(12;14) (q15;q24). Cancer Genetics, 204, 465–468. Kottickal, L.V., Sarada, B., Ashar, H., Chada, K. & Nagarajan, L. (1998) Preferential expression of HMGI-C isoforms lacking the acidic carboxy terminal in human leukemia. Biochemical
Author’s contributions KBN, IP, JT and RR performed the research and/or wrote the paper. HSW and AT provided essential diagnostic information and critically revised the manuscript. FM and SH designed the study and revised the manuscript.
Conflict of interest The authors declare no competing financial conflict of interest.
Ethics The study was approved by the Regional Ethics Committee and the Norwegian Directorate of Health. Kaja B. Nyquist1,2,3 Ioannis Panagopoulos1,2 Jim Thorsen1,2 Roberta Roberto1,2 Hilde S. Wik4 Anne Tierens5 Sverre Heim1,2,3 Francesca Micci1,2 1
Section for Cancer Cytogenetics, Institute for Medical Informatics, The
Norwegian Radium Hospital, Oslo University Hospital, 2Centre for Cancer Biomedicine, University of Oslo, 3Faculty of Medicine, University of Oslo, 4Department of Haematology, Oslo University Hospital, 5Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway E-mail:
[email protected]
Keywords: acute myeloid leukaemia, HMGA2, t(12;13)(q14;q31), cytogenetics, gene deregulation First published online 7 March 2012 doi: 10.1111/j.1365-2141.2012.09081.x
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