Overexpression of <i>HOXB7</i> and homeobox genes characterizes multiple myeloma patients lacking the major primary immunoglobulin heavy chain locus translocations
Authors: Luca Agnelli, Paola Storti, Katia Todoerti, Gabriella Sammarelli, Benedetta Dalla Palma, Marina Bolzoni, Alberto Rocci, Francesco Piazza, Gianpietro Semenzato, Antonio Palumbo, Antonino Neri, Nicola Giuliani
Journal: American Journal of Hematology (2011)

Homeobox (HOX) gene transcription factors are frequently deregulated in hematologic malignancies and involved in leukemogenic transformation [1]. Moreover, their overexpression has been associated with tumoral-induced neoangiogenesis in solid cancer [2]. The expression and the role of these genes have not yet been completely elucidated in multiple myeloma (MM). Recently, we reported that a small fraction of MM patients shows a HOXB7 overexpression as compared with normal samples and that HOXB7 expression correlates with bone marrow angiogenesis and the production of the proangiogenic factors by MM cells [3]. Other authors previously reported that HOXA cluster genes are expressed in a small fraction of MM patients [4]. Herein, we extended our previous evidences with the evaluation of the expression level of HOXB7 and the other gene family members in a large number of primary MM cells in relationship with the different molecular subgroups of MM and the presence of specific chromosome translocations. We found that HOXB7 and other genes of HOX family have a preferential distribution based on the characteristics of molecular MM subtypes based on the translocations/cyclins (TC) classification, suggesting a potential relationship between HOX genes expression, angiogenesis, and molecular features of MM patients. In this study, we analyzed HOXB7 and HOX cluster genes in a proprietary dataset including 132 samples profiled on GeneChip® HG-U133A arrays (available at NCBI Gene Expression Omnibus repository under accession #GSE13591) and stratified according the proposed TC classification [5]. Interestingly, a preferential distribution based on the characteristics of TC-based molecular MM subtypes was observed, being HOXB7 significantly upregulated (P = 1.077 e−10) in those cases that did not show any of the major primary IGH translocations (TC2 and TC3 groups, Fig. 1A). These data were confirmed in three additional publicly available datasets profiled on HG-U133A arrays: specifically, a similar behavior was observed in the MM samples at diagnosis from Dickens et al. [6] (GEO series GSE15695, Fig. 1B, P = 1.887 e−11), from Chng et al. [7] (GSE6477, Fig. 1C, P = 2.556 e−03), and from Mulligan et al. [8] (GSE9782, Fig. 1D, P = 5.949 e−10) datasets, encompassing a total number of 768 samples. Additionally, we evaluated the expression levels of HOXB7 in the 414 samples stratified into seven molecular MM subtypes described by Zhan et al. in their dataset [9] and further confirmed a significantly lower expression level (GSE2658, Fig. 1E, P << 1 e−10) in CD-1, CD-2, MS, and MF groups (showing common IGH translocations) compared with other subtypes (Fig. 1B). The occurrence of IGH translocated cases within HY, LB, and PR groups was negligible, if not null. Boxplot representation of HOXB7 expression level in GSE13591 (A), GSE15695 (B), GSE6477 (C), GSE9782 (D), and GSE2658 (E) datasets available at http://www.ncbi.nlm.nih.gov/geo/. From (A) to (D), we referred to TC groups previously described [<xref>5</xref>]. TC1: patients characterized by the t(11;14) or t(6;14) translocation; TC2: patients showing low to moderate levels of the CCND1 gene in the absence of any primary IGH translocation; TC3: patients that do not fall into any of the other groups; TC4: patients showing high CCND2 levels and the presence of the t(4;14) translocation; TC5: patients with either the t(14;16) or the t(14;20) translocation. In (E), MM patients were stratified according seven-group classification proposed by Zhan et al. [9]. HY: hyperdiploid patients; LB: low bone disease patients; PR: patients overexpressing proliferation genes; MS: patients overexpressing MMSET genes as a consequence of t(4;14); MF: patients overexpressing MAF/MAFB as a consequence of t(14;16) or t(14;20), respectively; patients overexpressing CD-1 and CD-2: CCND1/CCND3 Groups 1 and 2 as a consequence of t(11;14) or t(6;14), respectively. On the basis of the structure similarities of the HOX domain-containing transcription factors, we additionally investigated the expression patterns of other HOX genes and their correlation with molecular subtypes in MM. Of note, we found that the expression levels of several members of the HOXA and HOXB gene families together with MEIS1 and MEIS2 showed a globally positive correlation in the proprietary dataset (Fig. 2A); some of these genes, including HOXA3, HOXA5, HOXA10, MEIS2, and HOXB4, were also significantly upregulated in cases without primary IGH translocations. (A) Correlation matrix for 21 HOX genes based on the expression values in the 132 MM cases of the proprietary GSE13591 dataset. The color scale bar ranges from −1 (blue, negative correlation) to 1 (red, positive correlation). (B) Boxplot representation of the expression values of 11 HOX transcripts as assessed by Q-RT-PCR (RT2 profiler PCR array customized, SABiosciences, Frederick, MD). P < 0.05 in the comparisons of the expression values in TC2/TC3 with those in TC1/TC4/TC5 cases are indicated with asterisks. Thus, we validated by quantitative real time (RT)-PCR the expression of HOXB7 and other 10 HOX transcripts in an independent cohort of 24 primary purified CD138+ MM samples collected at diagnosis. All the samples were characterized by fluorescence in situ hybridization (FISH) analysis for the major translocations and grouped according to the TC classification by gene expression profiling. Our data revealed a significant upregulation of HOXA3 (P = 0.0237), HOXB7 (P = 3 e−05), HOXA5 (P = 0.0046), MEIS2 (P = 0.0066), HOXA10 (P = 0.0152), HOXB4 (P = 0.0121) in patients without t(4;14), t(11;14), or MAF family genes translocations (Fig. 2B). To further support these data, no specific correlation was found between HOXB7 and the other HOX genes either with 13q deletion, or 1q gain aberrations, or hyperdiploidy. It is worth noting that, although not statistically significant, the only three samples overexpressing HOXA9 were included in TC2/TC3 group not carrying known IGH translocations, in accordance with recent observations in a dataset of MM cases analyzed using whole-exome sequencing approach [10]. In the same paper, Chapman et al. evidenced that HOXA9-depleted cells exhibited a competitive disadvantage in a large number of myeloma cell lines and suggested its role as a putative new therapeutic target. Overall, these data indicate that HOXB7 and related HOX genes are upregulated in specific subsets of MM patients lacking the major IGH translocations, irrespectively of hyperdiploidy or other known cytogenetic lesions. Together with our previous findings [3], this suggests a potential relationship between HOX genes expression, angiogenesis and molecular features of MM patients with possible therapeutic implications, and prompts for a comprehensive evaluation in the context of tumor microenvironment. Gene expression profiling analysis and statistical analysis. Expression values were generated from intensity signals using the RMA algorithm and GeneAnnot custom chip definition files, as previously described [11]; values have been background adjusted, normalized using quantile normalization, and expression measure calculated using median polish-summarization. In the case of GSE2658, which lacks raw data, the very same normalized data described in Zhan et al. and deposited on GEO repository were used for calculations. Kruskal–Wallis one-way analysis of variance by ranks or Wilcoxon rank sum tests was used for testing differences in the expression values distribution among groups, where appropriate. Kendall's tau coefficient was used to test correlation between the expression levels of the investigated genes. All calculations were performed using appropriate function in Rsoftware. CD138+ plasma cells purification from MM patients. CD138+ plasma cells were purified by bone marrow aspirates (5 mL) obtained from iliac crest of 24 consecutive patients (median age 66 years; range 53–73) with newly diagnosed MM (ISS Stages I–III) by an immunomagnetic method using anti-CD138 mAb-coated microbeads (MACS, Miltenyi Biotec, Bergisch-Gladbach, Germany). Only samples with purity >90% checked by flow cytometry (FACScan, Becton Dickinson; San Jose, CA) were tested. Quantitative RT-PCR. RT-PCR was performed by adding 2 out of 20 μL of cDNA, obtained from 1 μg of RNA, to an universal master Mix (Applied Biosystem, Life Technologies, Carlsbad, CA), primers and TaqMan probes. Primers and probes for HOXB7, HOXB4, HOXB5, HOXB8, HOXA3, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, and MEIS2 were synthesized and purchased from Applied Biosystems. FISH analysis. Interphase FISH analysis was performed as previously described [11] on CD138-purified plasma cells using probes for chromosomes 1q21, 13q14.3, and 17p13 and for the translocations t(11;14)(q13;q32.3), t(4;14)(p16.3;q32.3), and t(14;16)(q32.3;q23). Hybridization was performed according to the manufacturer's instructions (Vysis, Downers Grove, IL). Luca Agnelli*, Paola Storti , Katia Todoerti*, Gabriella Sammarelli , Benedetta Dalla Palma , Marina Bolzoni , Alberto Rocci , Francesco Piazza§, Gianpietro Semenzato§, Antonio Palumbo , Antonino Neri*, Nicola Giuliani , * Department of Medical Sciences, University of Milano and Hematology 1, CTMO – Centro Trapianti Midollo Osseo, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy, Department of Internal Medicine and Biomedical Science, Hematology and BMT Center, University of Parma, Parma, Italy, Department of Oncology, Hematology Section, University of Torino, Torino, Italy, § Department of Clinical and Experimental Medicine, University of Padova, Padova, Italy.