High-resolution genomic profiling of adenomas and carcinomas of the salivary glands reveals amplification, rearrangement, and fusion of HMGA2.

Carcinoma ex pleomorphic adenoma (Ca-ex-PA) is an epithelial malignancy developing within a benign salivary gland pleomorphic adenoma (PA). Here we have used genome-wide, high-resolution array-CGH, and fluorescence in situ hybridization to identify genes amplified in double min chromosomes and homogeneously staining regions in PA and Ca-ex-PA and to identify additional genomic imbalances characteristic of these tumor types. Ten of the 16 tumors analyzed showed amplification/gain of a 30-kb minimal common region, consisting of the 5'-part of HMGA2 (encoding the three DNA-binding domains). Coamplification of MDM2 was found in nine tumors. Five tumors had cryptic HMGA2-WIF1 gene fusions with amplification of the fusion oncogene in four tumors. Expression analysis of eight amplified candidate genes in 12q revealed that tumors with amplification/rearrangement of HMGA2 and MDM2 had significantly higher expression levels when compared with tumors without amplification. Analysis of individual HMGA2 exons showed that the expression of exons 3-5 were substantially reduced when compared with exons 1-2 in 9 of 10 tumors with HMGA2 activation, indicating that gene fusions and rearrangements of HMGA2 are common in tumors with amplification. In addition, recurrent amplifications/gains of 1q11-q32.1, 2p16.1-p12, 8q12.1, 8q22-24.1, and 20, and losses of 1p21.3-p21.1, 5q23.2-q31.2, 8p, 10q21.3, and 15q11.2 were identified. Collectively, our results identify HMGA2 and MDM2 as amplification targets in PA and Ca-ex-PA and suggest that amplification of 12q genes (in particular MDM2), deletions of 5q23.2-q31.2, gains of 8q12.1 (PLAG1) and 8q22.1-q24.1 (MYC), and amplification of ERBB2 may be of importance for malignant transformation of benign PA.

Frequent fusion of the CRTC1 and MAML2 genes in clear cell variants of cutaneous hidradenomas.

Fusion of the CREB regulated transcription coactivator CRTC1 (a.k.a. MECT1, TORC1, or WAMTP1) to the Notch coactivator MAML2 is a characteristic feature of low-grade mucoepidermoid carcinomas of salivary and bronchial glands. The CRTC1-MAML2 fusion protein acts by inducing transcription of cAMP/CREB target genes, and this activity is crucial for the transforming properties of the protein. Here we show that the CRTC1-MAML2 gene fusion is also frequent in benign hidradenomas of the skin. FISH and RT-PCR analyses revealed that hidradenomas are genetically heterogeneous, and that 10 of the 20 tumors analyzed (50%) contained the CRTC1-MAML2 gene fusion and expressed the resulting fusion transcript. Immunohistochemical analysis demonstrated expression of the fusion protein in the majority of tumor cells, including clear cells, poroid cells, and cells with epidermoid and ductal differentiation. In addition, we could show that all fusion-positive tumors were morphologically distinguished by the presence of more or less abundant areas of clear cells whereas all fusion-negative tumors lacked clear cells. Our findings thus demonstrate that the CRTC1-MAML2 gene fusion is frequent in hidradenomas and is associated with clear cell variants of this tumor. Taken together, the present and previous observations indicate that the CRTC1-MAML2 fusion is etiologically linked to benign and low-grade malignant tumors originating from diverse exocrine glands rather than being linked to a separate tumor entity.

Molecular genetic analyses of the TMPRSS2-ERG and TMPRSS2-ETV1 gene fusions in 50 cases of prostate cancer.

Recently, gene fusions between the androgen responsive gene TMPRSS2 and members of the ETS-family of DNA-binding transcription factor genes were found in prostate cancer. Recurrent fusions were identified between the 5'-noncoding region of TMPRSS2 and ERG, or less frequently ETV1 or ETV4, resulting in overexpression of normal or truncated ETS-proteins. Herein, we have analyzed a series of 50 prostate cancer samples for expression of TPRSS2-ERG and TMPRSS2-ETV1 fusion transcripts. RT-PCR analysis revealed TMPRSS2-ERG fusion transcripts in 18 of the 50 tumors (36%). None of the tumors expressed a TMPRSS2-ETV1 fusion. Our findings show that the TMPRSS2-ERG fusion is common in prostate cancer and that the related TMPRSS2-ETV1 fusion is very rare. However, the frequency of ERG-fusions in the present study is somewhat lower than previously observed, indicating heterogeneity with regard to expression of ETS-gene fusions in subsets of prostate cancers. Moreover, clinical follow-up studies showed a clear tendency that fusion-positive tumors were associated with lower Gleason grade and better survival than fusion-negative tumors. Our findings suggest that ERG gene fusions might be of prognostic significance in prostate cancer.

Analysis of chromosome 10 aberrations in rat endometrial cancer-evidence for a tumor suppressor locus distal to Tp53.

We have recently shown in the BDII rat model of human endometrial adenocarcinoma (EAC), rat chromosome 10 (RNO10) is frequently involved in chromosomal aberrations. In the present study, we investigated the association between RNO10 deletions, allelic imbalance (AI) at RNO10q24 and Tp53 mutation in 27 rat EAC tumors. We detected chromosomal breakage accompanied by loss of proximal and/or gain of distal parts of RNO10 in approximately 2/3 of the tumors. This finding is suggestive of a tumor suppressor activity encoded from the proximal RNO10. Given the fact that Tp53 is located at RNO10q24-q25, we then performed Tp53 mutation analysis. However, we could not find a strong correlation between AI/deletions at RNO10q24 and Tp53 mutation. Instead, the observed patterns for AI, chromosomal breaks and deletions suggest that major selection was directed against a region located close to, but distal of Tp53. In different human malignancies a similar situation of AI at chromosome band 17p13.3 (HSA17p13.3) unassociated with TP53 mutation has been observed. Although RNO10 is largely homologous to HSA17, the conservation with respect to gene order among them is not extensive. We utilized publicly available draft DNA sequences to study intrachromosomal rearrangement during the divergence between HSA17 and RNO10. By using reciprocal comparison of rat and human genome data, we could substantially narrow down the candidate tumor suppressor region in rat from 3 Mb to a chromosomal segment of about 0.5 Mb in size. These results provide scientific groundwork for identification of the putative tumor suppressor gene(s) at 17p13.3 in human tumors.

Molecular classification of mucoepidermoid carcinomas-prognostic significance of the MECT1-MAML2 fusion oncogene.

Mucoepidermoid carcinomas (MECs) of the salivary and bronchial glands are characterized by a recurrent t(11;19)(q21;p13) translocation resulting in a MECT1-MAML2 fusion in which the CREB-binding domain of the CREB coactivator MECT1 (also known as CRTC1, TORC1 or WAMTP1) is fused to the transactivation domain of the Notch coactivator MAML2. To gain further insights into the molecular pathogenesis of MECs, we cytogenetically and molecularly characterized a series of 29 MECs. A t(11;19) and/or an MECT1-MAML2 fusion was detected in more than 55% of the tumors. Several cases with cryptic rearrangements that resulted in gene fusions were detected. In fusion-negative MECs, the most common aberration was a single or multiple trisomies. Western blot and immunohistochemical studies demonstrated that the MECT1-MAML2 fusion protein was expressed in all MEC-specific cell types. In addition, cotransfection experiments showed that the fusion protein colocalized with CREB in homogeneously distributed nuclear granules. Analyses of potential downstream targets of the fusion revealed differential expression of the cAMP/CREB (FLT1 and NR4A2) and Notch (HES1 and HES5) target genes in fusion-positive and fusion-negative MECs. Moreover, clinical follow-up studies revealed that fusion-positive patients had a significantly lower risk of local recurrence, metastases, or tumor-related death compared to fusion-negative patients (P = 0.0012). When considering tumor-related deaths only, the estimated median survival for fusion-positive patients was greater than 10 years compared to 1.6 years for fusion-negative patients. These findings suggest that molecularly classifying MECs on the basis of an MECT1-MAML2 fusion is histopathologically and clinically relevant and that the fusion is a useful marker in predicting the biological behavior of MECs.

Clear cell hidradenoma of the skin-a third tumor type with a t(11;19)--associated TORC1-MAML2 gene fusion.

Recent studies have shown that the t(11;19)(q21;p13) translocation in mucoepidermoid carcinomas and benign Warthin's tumors results in a fusion of the N-terminal CREB-binding domain of the cAMP coactivator TORC1 (a.k.a. MECT1 and WAMTP1) to the Notch coactivator MAML2. Here we show that a third tumor type, clear cell hidradenoma of the skin, also expresses this gene fusion. RT-PCR analysis of a clear cell hidradenoma with a t(11;19)(q21;p13) translocation revealed expression of a TORC1-MAML2 fusion transcript consisting of exon 1 of TORC1 fused to exons 2-5 of MAML2. Because the fusion was only detected in a single case, the frequency of this aberration in clear cell hidradenomas remains unknown. These results demonstrate that the t(11;19) in mucoepidermoid carcinoma, Warthin's tumor, and clear cell hidradenoma targets the same genes and results in identical gene fusions, indicating that at least subgroups of these glandular tumors evolve through activation of the same molecular pathways.