Leukemia (2013) 27, 92–99 & 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13 www.nature.com/leu ORIGINAL ARTICLE Immunoglobulin gamma heavy chain gene with somatic hypermutation is frequently expressed in acute myeloid leukemia X Qiu1,2, X Sun3,ZHe1, J Huang2,FHu2, L Chen4, P Lin1,MJYou1, LJ Medeiros1 and CC Yin1 Expression of immunoglobulin (Ig), a marker characteristic of B cells, has been reported in epithelial cells and has been suggested to have a role in their survival and growth. We assessed the frequency and level of Ig gamma heavy chain (IgG) expression in acute myeloid leukemia (AML), and found that IgG was expressed at a high frequency and level in AML cell lines and primary myeloblasts, but not in monocytes or neutrophils from patients with non-hematopoietic neoplasms or healthy controls. AML-derived IgG had the same molecular weight as B cell-derived IgG and was secreted. We further detected IgG VHDJH transcripts in AML cell lines and sorted primary myeloblasts, confirming that IgG expression was indeed produced by AML cells. AML-derived IgG gene rearrangements showed evidence of somatic hypermutation of the variable (V) gene segments, and restricted (AML cell lines) or biased (primary myeloblasts) V usage. Anti-human IgG reduced cell viability and induced apoptosis in AML cell lines. Although the function of the AML-derived IgG is unclear, our findings suggest that AML-derived IgG may be a novel AML-related gene that contributes to leukemogenesis and AML progression. AML-derived IgG may serve as a useful molecular marker for monitoring minimal residual disease or designing target therapy. Leukemia (2013) 27, 92–99; doi:10.1038/leu.2012.184 Keywords: IgG; acute myeloid leukemia; VHDJH gene rearrangements INTRODUCTION expression between these cells that might have clinical utility. Our Immunoglobulin (Ig) was previously thought to be produced only results show for the first time that IgG is expressed and secreted in by B lymphocytes and plasma cells. During the past decade, AML cell lines and primary myeloblasts at a high frequency and however, this concept has been challenged by a series of studies. level. We also found that IgG VHDJH was rearranged only in AML Different types of Ig, including IgG, IgM and IgA, have been shown cells, but not in monocytes or neutrophils. Furthermore, the AML- to be expressed in many types of neoplastic epithelial cells, derived IgG Variable (V) region revealed somatic hypermutation including those arising in the breast, colon, lung, liver, neck and and a restricted or biased usage of certain V segments. Preliminary mouth. In these cells, Ig is present in both membranous and functional study showed that anti-human IgG reduced cell viability secreted forms, similar to B cell-derived Ig.1–6 Subsequently, Ig and induced apoptosis in AML cell lines in culture. These results expression has been detected in several types of non- suggest that AML-derived IgG may have a role in AML patho- hematopoietic cells from healthy individuals, including epithelial genesis and progression, and potentially could serve as a novel cells, germ cells, neurons and myocardial cells.7–9 In addition, Ig molecular marker for the monitoring of minimal residual disease expressed in these normal cells shows some unique and development of target therapy. characteristics, including restricted usage of certain sequences,9,10 a unique glycosylation profile11,12 and different regulatory mechanisms of gene expression than are known in B MATERIALS AND METHODS cells.13,14 Moreover, it has been shown recently that non- AML and B-cell lymphoma cell lines hematopoietic cell-derived Ig is involved in cell survival and We used the AML cell lines, HEL, HL-60, KG-1, NB4, THP-1 and OCI-AML3, and proliferation.1,5 These findings suggest that non-hematopoietic the B-cell lymphoma cell line, SP53 (courtesy of Dr M Wang, Department of Myeloma, UT MD Anderson Cancer Center). All cell lines were maintained in cell-derived Ig is a novel molecule with a unique structure and RPMI-1640 medium (Life Technologies Inc., Carlsbad, CA, USA) supplemented function that may have a role in carcinogenesis. with 10% fetal bovine serum (HyClone/Thermal Scientific, Logan, UT, USA) Myeloid cells originate from hematopoietic stem cells in the and 2 mML-glutamine at 37 1C with humidified 5% CO2. bone marrow. It has been believed that myeloid cells cannot produce Ig. In this study, we sought to explore whether acute Patient samples myeloid leukemia (AML) cells (myeloblasts) as well as other cells of The study was conducted according to an institutional review board- myeloid origin, that is, mature normal monocytes and neutrophils, approved laboratory protocol. Peripheral blood specimens from 14 AML could produce Ig. We also sought to determine differences in Ig patients were analyzed for expression of IgG on the cell membrane of 1Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 2Department of Immunology, School of Basic Medical Sciences, Peking University, Beijing, China; 3Department of Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA and 4Department of Pathology, The University of Texas-Medical School, Houston, TX, USA. Correspondence: Dr CC Yin, Department of Hematopathology, UT MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 72, Houston, TX 77030, USA. E-mail: [email protected] or Dr X Qiu, Department of Immunology, School of Basic Medical Sciences, Peking University, Beijing 100191, China. E-mail: [email protected] Received 27 April 2012; revised 18 June 2012; accepted 2 July 2012; accepted article preview 9 July 2012; advance online publication, 14 August 2012 IgG gene expression in acute myeloid leukemia X Qiu et al 93 þ þ CD33 cells. To analyze IgG VHDJH gene transcription in CD33 AML cells, IgG-HRP (1:2000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) as the we assessed peripheral blood specimens from another 15 AML patients. secondary antibody, and a chemiluminescent substrate (Mabtech, þ We also analyzed IgG VHDJH gene transcription in CD33 adult monocytes Mariemont, OH, USA). and neutrophils derived from peripheral blood specimens of 15 patients with non-hematopoietic neoplasms as well as 10 healthy individuals. The Enzyme-linked immunosorbent assay clinicopathologic data were obtained by review of medical records. Approximately 2 Â 105 cells from AML cell lines or AML patients were plated per well in six-well plates, and cultured for 0, 24, 48, 72 or 96 h. The Molecular genetic analyses culture supernatant was collected, and secreted IgG was detected using an Karyotypic analysis was performed on metaphase cells prepared from ELISA kit for human IgG (Mabtech) according to manufacturer’s bone marrow aspirate specimens using standard techniques.15 The results instructions. were reported using the 2005 International System for Human Cytogenetic Nomenclature. Detection of VHDJH transcripts by reverse transcription-PCR Genomic DNA extracted from bone marrow aspirate specimens was Total RNA was extracted from AML cell lines using Trizol reagent amplified by PCR and subjected to mutational analysis for codons 12, 13 or (Invitrogen, Carlsbad, CA, USA). For sorted CD33 þ CD19 À CD138 À or 61 of KRAS and NRAS by pyrosequencing using a PSQ HS 96 Pysosequencer CD33 À CD19 þ CD138 À cells, total RNA was extracted using the RNeasy (Biotage, Uppsala, Sweden),16 or KIT (exons 8 and 17) and CEBPA by direct Micro Kit (Qiagen Inc., Chatsworth, CA, USA) according to manufacturer’s Sanger sequencing on an ABI Prism 3100 Genetic Analyzer (Applied instructions. Reverse transcription (RT) was performed using the Sensiscript Biosystems, Foster City, CA, USA).16,17 A fluorescence-based multiplex PCR RT Kit (Qiagen Inc.). IgG V DJ transcripts were detected by a nested PCR. followed by capillary electrophoresis was used to detect internal tandem H H The primers and conditions for the first-round PCR for the amplification of duplication and D835 point mutation of the FLT3 gene and exon 12 V DJ were described previously.10 For the second-round PCR, an mutation of the NPM1 gene using genomic DNA and an ABI Prism 3100 H H upstream primer that anneals to the framework 2 (FR2) region (FR2: Genetic Analyzer.18,19 50-TGG(A/G)TCCG(A/C/G)CAG(G/C)C(T/C)CC(A/C/G/T)GG-30), coupled with a JH primer (JH: 50-AACTGCAGAGGAGACGGTGACC-30), was used.20 The Flourescence-activated cell sorting amplification products were gel-purified using a DNA purification column For sorting of myeloblasts from AML patients, mononuclear cells were (Qiagen Inc.), ligated into the pCR2.1 vector (Invitrogen), and transformed isolated from peripheral blood by Ficoll-Hypaque density gradient into DH5a-competent bacteria (Invitrogen). In all, 5–10 colonies per sample were chosen randomly, and sequenced using the T7 primer (Invitrogen). centrifugation, and washed with 0.01M PBS. The mononuclear cells were stained with monoclonal antibodies specific to CD19 (APC, BD Pharmigen, San Diego, CA, USA), CD33 (FITC, BD Pharmigen) and CD138 (PE, BD Analysis of rearranged IgG VHDJH genes Pharmigen) at 4 1C for 15 min. After washing twice with PBS, the cells were The rearranged V DJ sequences were compared with those in the basic sorted using a FACScan instrument (BD Pharmigen). CD33 þ CD19 À H H À À þ local alignment search tool (BLAST, http://www.ncbi.nlm.nih.gov/BLAST) CD138 cells were selected. CD33 CD19 cells also were collected and and Immunogenetics (http://www.imgt.org/) databases to identify the best used as a positive control. Anti-mouse IgG1-PE was used as an isotype control. matching germline gene segments and junctions. For the analysis of For sorting of monocytes and neutrophils, erythrocytes from patients somatic hypermutation, the part of the IgG V region from FR2 to JH was with non-hematopoietic neoplasms and healthy individuals were lysed by used.