Deregulation of HMGA2 in an aggressive angiomyxoma with t(11;12)(q23;q15)
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- Micci, F., Panagopoulos, I., Bjerkehagen, B. et al. Virchows Arch (2006) 448: 838. doi:10.1007/s00428-006-0186-5
Aggressive angiomyxoma is a soft-tissue neoplasm with a predilection for the pelvic and perineal regions and a tendency to recur locally. Cytogenetic data on this tumor type are limited to five cases, three of which showed rearrangement of chromosomal bands 12q13–15. Molecular investigation of two of the tumors identified the HMGA2 gene as the target of the 12q rearrangements. However, the two previously analyzed tumors were different at the molecular level: in one, the rearrangement of 12q13–15 resulted in a fusion product, whereas, in the second case, the breakpoint was telomeric (3′) to the HMGA2, leaving the gene intact although expressed in its entire length. To shed more light on the pathobiology of aggressive angiomyxoma and to investigate the molecular mechanisms behind the involvement of the HMGA2 gene in this tumor type (fusion transcript vs deregulated expression), we investigated, cytogenetically and with molecular techniques, one such tumor which presented a t(11;12)(q23;q15) as the sole karyotypic aberration. FISH analyses demonstrated no structural alteration of HMGA2 at the cytogenetic level; however, expression of the full-length gene was detected molecularly.
KeywordsAggressive angiomyxomaCytogenetic HMGA2
Aggressive angiomyxoma is an infiltrative mesenchymal tumor composed of fibroblasts, myofibroblasts, and numerous thick-walled blood vessels embedded in an abundant myxoid matrix. It occurs in adult women during reproductive years. In addition to its location in the external genitalia of women, cases have also been reported in men, where they occur in the perineal/perianal region [3, 8, 33]. Although the tumor is locally aggressive and prone to recur after surgical removal, metastases are exceedingly rare [10, 20, 23, 34].
Cytogenetic studies have revealed clonal chromosomal abnormalities in five aggressive angiomyxomas. Four tumors had abnormalities involving chromosome 12, including one case with monosomy 12 among other changes  and three cases with rearrangements of chromosomal bands 12q13–15 [4, 15, 24]. The fifth tumor showed monosomy of the X chromosome as the sole karyotypic aberration . Molecular investigations of two cases with structural rearrangements of 12q13–15 have identified the high-mobility-group (HMG) protein gene, HMGA2, previously known as HMGIC, as the target gene [14, 24]. Its involvement differed between the two analyzed tumors, however. The 12q15 rearrangement in an aggressive angiomyxoma with the karyotype 46,XX,der(5)t(5;12)(q31;p11),der(12)inv(12)(p11q15)t(5;12)(q31;p11) resulted in a fusion product containing the first three exons of HMGA2 followed by a novel 317 bp sequence mapping to 12p11 . The second molecularly examined tumor, with the karyotype 46,XX,t(8;12)(p12;q15), had a breakpoint telomeric (3′) of HMGA2, leaving the gene intact but deregulated and expressed.
To shed more light on the pathobiology of aggressive angiomyxoma and, in particular, to investigate the molecular mechanisms behind the involvement of the HMGA2 gene in this tumor type (fusion transcript vs deregulated expression), we examined using molecular techniques another such tumor which presented a t(11;12)(q23;q15) as the sole karyotypic aberration.
Materials and methods
The specimens intended for cytogenetic analysis were mechanically and enzymatically disaggregated and short-term-cultured according to Mandahl . Chromosome preparations were made and G-banded using Wright stain. The subsequent cytogenetic analysis and karyotypic description followed the recommendations of the ISCN . Because no metaphase spreads were available for fluorescence in situ hybridization (FISH) analysis, interphase nuclei were used to check for possible rearrangement of the HMGA2 gene. A pool of cosmid clones spanning the HMGA2 breakpoint region, 142H1, 245E8, and 154F9 containing exons 1–2 and 156A4 and 27E12 containing exons 4–5, was selected . The clones were grown in selective media, and DNA was extracted according to standard methods . DNA probes were directly labeled with Cy3-dCTP (Amersham, Buckinghamshire, UK) and indirectly with Biotin-dUTP (Molecular Probes, Invitrogen, Carlsbad, CA, USA) by nick-translation, and detected with streptavidin-diethylaminocoumarin (DEAC; Invitrogen). The subsequent hybridization conditions as well as the detection procedure were according to standard protocols . The hybridization was analyzed using a CytoVision system (Applied Imaging, Newcastle, UK).
Molecular genetic investigations
Primers used for PCRs of HMGA2
cDNA was synthesized using 5 μg of total RNA in 20 μl reaction mixture [50 mM Tris–HCl pH 8.3 (at 25°C), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol (DTT), 1 mMol each dNTP, 20 U RNAse inhibitor (RNA guard, Amersham Biosciences, Piscataway, USA), 0.5 pM random hexamers, and 400 U M-MLV Reverse Transcriptase (Invitrogen)]. The reaction was carried out at 37°C for 60 min, heated for 5 min at 65°C, and then kept at 4°C until analysis. As an internal control, cDNA quality was checked using ABL1-specific primers .
The PCR amplification was performed using 1 μl of the cDNA as template in 50 μl reaction volume containing 20 mM Tris–HCl pH 8.4 (at 25°C), 50 KCl, 1.25 mM MgCl2, 0.2 mM of each dNTP, 1 U Platinum Taq polymerase (Invitrogen), and 0.5 μM of each of the forward and reverse primers used. The HMGA2 transcripts were amplified using an initial denaturation for 5 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 59°C, and 1 min at 72°C and a final extension for 5 min at 72°C. The PCR products were analyzed by electrophoresis through 1.2% agarose gel stained with ethidium bromide.
Based on the observation that chromosomal bands 12q13–15 are nonrandomly involved in structural abnormalities in aggressive angiomyxoma, they have been found rearranged in three out of five previously analyzed tumors, and that HMGA2 was the target gene in the only two examined tumors having such rearrangements, we decided to assess the status of HMGA2 in an aggressive angiomyxoma showing a t(11;12) as the sole karyotypic abnormality. FISH analyses detected no structural alteration of HMGA2, but PCR analysis of the gene using a specific primer combination for exons 1–3 and 1–5 revealed that it was expressed with an intact, full-length product. Expression of the entire HMGA2 gene has been shown to be achieved through alterations affecting 5′ regulatory elements or the 3′ untranslated region, leading to a stabilized mRNA in uterine leiomyomata and transfected HeLa cells [6, 26, 29].
As we only analyzed interphase nuclei by FISH, we could not determine the exact position of the breakpoint on 12q, whether it was 5′ or 3′ of the HMGA2 gene, and, therefore, we could not identify which mechanism led to a stable HMGA2 mRNA in our sample, i.e., alteration of the 5′ regulatory elements or disruption of the 3′ untranslated region. However, FISH data on the previously reported aggressive angiomyxoma showing a full-length HMGA2 transcript placed the breakpoint 3′ (telomeric) to HMGA2 . An alternative activation mechanism for HMGA2 was reported by Kazmierczak et al.  in an aggressive angiomyxoma with inv(12)(p11.2q15); in that tumor, the third exon of HMGA2 was fused with a 317-bp ectopic sequence from 12p. Apparently, both main molecular mechanisms of HMGA2 involvement, the generation of a fusion transcript and deregulation of expression, may be operative in aggressive angiomyxoma [14, 24].
This variability in how activation of HMGA2 is achieved is similar to what has been found in other benign mesenchymal tumors showing involvement of this gene. Both in bone and soft tissue chondromas  and uterine leiomyomata , the transcriptional activation may occur via alternative mechanisms. It is also noteworthy that some tumors apparently express both full-length and truncated HMGA2 transcript [9, 32]. In the aggressive angiomyxoma with a inv(12)(p11q15) resulting in a HMGA2 fusion product reported by Kazmierczak et al. , the novel sequence was not homologous to any known gene or expressed-sequence tag, and theoretically contributed only seven amino acid residues before the in-frame stop codon, effectively resulting in a truncated HMGA2 protein containing just the three DNA-binding domains.
The HMGA2 gene encodes a member of the high-mobility group A (HMGA) of small, non-histonic, chromatin-associated proteins. These proteins are believed to affect transcription as architectural elements by bending the DNA and by interacting with a large number of other proteins, mainly transcription factors, but also to influence chromatin changes during the cell cycle . The HMGA2 gene is highly expressed in embryonic tissues but has in adults only been found expressed in kidney, lung, and synovia [7, 11]. It has nevertheless been predicted to play a role in adult tissues as well . Experiments on mice demonstrated the importance of the HMGA2 protein for embryonic cell growth [2, 35]. Recently, Ligon et al.  reported an 8-year-old boy with a constitutional de novo chromosomal inversion resulting in disruption of HMGA2 and aberrant adiposity and somatic overgrowth. This phenotype, particularly with respect to overgrowth and lipogenesis, is strikingly similar to that of murine models expressing truncated Hmga2 transcripts .
HMGA2 has been found rearranged in a variety of solid tumors including lipoma, uterine leiomyoma, chondroma, and pulmonary chondroid hamartoma [1, 9, 22, 26, 29]. It recombines with other chromosomal sites through translocations, insertions, and inversions. The involvement of HMGA2 in different mesenchymal tumors and the promiscuous nature of the translocations involving this gene are reminiscent of that of another gene encoding a nuclear scaffold protein associated with neoplasia. The mixed lineage leukemia (MLL) gene is rearranged in numerous acute lymphoid and myeloid leukemias in humans and more than 40 different partner genes have been identified . MLL contains three motifs located near its amino terminus that are highly homologous to the core AT hook DNA-binding domains of the HMGA2 protein; however, whether the HMGA proteins and MLL share any common mechanism of transformation remains to be shown.
Aggressive angiomyxomas are infiltrative tumors with a marked tendency to recur locally. Immunostaining and ultrastructural studies indicate that this neoplasm derives from a primitive mesenchymal cell normally found in the lower female genital tract and capable of myofibroblastic differentiation. Because the most common pattern of behavior of this tumor involves no metastasis, aggressive angiomyxoma has been considered invariably benign [10, 20, 23]. However, two cases of aggressive angiomyxoma with metastasis leading to the patients’ death [5, 31] highlight the need to consider it an intermediate tumor with unpredictable and sometimes unfavorable behavior . On the other hand, as HMGA2 has hitherto only been found to be pathogenetically involved in benign mesenchymal tumors [1, 9, 22, 26, 29], the involvement of the same gene in aggressive angiomyxoma favors its classification as a benign neoplasm. In addition, as normal tissues adjacent to aggressive angiomyxomas are negative for HMGA2 expression, inappropriate HMGA2 expression may potentially be useful as a marker of microscopic residual disease using the immunoperoxidase technique with an anti-HMGA2 antibody [20, 23].
This work was supported by grants from The Norwegian Cancer Society, COST Action B-19—Molecular cytogenetics of solid tumors—Short-term Scientific Mission programme, and The Gunnar Nilsson’s Cancer Foundation.