Salivary acinic cell carcinoma: reappraisal and update.

Epidemiologic and clinicopathologic features, therapeutic strategies, and prognosis for acinic cell carcinoma of the major and minor salivary glands are critically reviewed. We explore histopathologic, histochemical, electron microscopic and immunohistochemical aspects and discuss histologic grading, histogenesis, animal models, and genetic events. In the context of possible diagnostic difficulties, the relationship to mammary analog secretory carcinoma is probed and a classification is suggested. Areas of controversy or uncertainty, which may benefit from further investigations, are also highlighted.

EWS and ATF-1 gene fusion induced by t(12;22) translocation in malignant melanoma of soft parts.

The genes involved in the t(12;22)(q13;q12) translocation found recurrently in malignant melanoma of soft parts have been characterized and shown to form, in four cases studied, hybrid transcripts. The deduced chimaeric protein encoded by the der(22) chromosome consists of the N-terminal domain of EWS linked to the bZIP domain of ATF-1, a transcription factor which may normally be regulated by cAMP. ATF-1 has not previously been implicated in oncogenesis. EWS was first identified as forming a hybrid transcript in Ewing's sarcoma, which links its N-terminal domain to the DNA binding domain of the FLI-1 gene. Thus the oncogenic conversion of EWS follows a common scheme of activation, exchanging its putative RNA binding domain with different DNA binding domains that appear to be tumour-specific.

Promoter swapping between the genes for a novel zinc finger protein and beta-catenin in pleiomorphic adenomas with t(3;8)(p21;q12) translocations.

Pleiomorphic adenoma of the salivary glands is a benign epithelial tumour occurring primarily in the major and minor salivary glands. It is by far the most common type of salivary gland tumour. Microscopically, pleiomorphic adenomas show a marked histological diversity with epithelial, myoepithelial and mesenchymal components in a variety of patterns. In addition to a cytogenetic subgroup with normal karyotypes, pleiomorphic adenomas are characterized by recurrent chromosome rearrangements, particularly reciprocal translocations, with breakpoints at 8q12, 3p21, and 12q13-15, in that order of frequency. The most common abnormality is a reciprocal t(3;8)(p21;q12). We here demonstrate that the t(3;8)(p21;q12) results in promoter swapping between PLAG1, a novel, developmentally regulated zinc finger gene at 8q12, and the constitutively expressed gene for beta-catenin (CTNNB1), a protein interface functioning in the WG/WNT signalling pathway and specification of cell fate during embryogenesis. Fusions occur in the 5'-non-coding regions of both genes, exchanging regulatory control elements while preserving the coding sequences. Due to the t(3;8)(p21;q12), PLAG1 is activated and expression levels of CTNNB1 are reduced. Activation of PLAG1 was also observed in an adenoma with a variant translocation t(8;15)(q12;q14). Our results indicate that PLAG1 activation due to promoter swapping is a crucial event in salivary gland tumourigenesis.

Exclusive paternal origin of new mutations in Apert syndrome.

Apert syndrome results from one or other of two specific nucleotide substitutions, both C-->G transversions, in the fibroblast growth factor receptor 2 (FGFR2) gene. The frequency of new mutations, estimated as 1 per 65,000 live births, implies germline transversion rates at these two positions are currently the highest known in the human genome. Using a novel application of the amplification refractory mutation system (ARMS), we have determined the parental origin of the new mutation in 57 Apert families: in every case, the mutation arose from the father. This identifies the biological basis of the paternal age effect for new mutations previously suggested for this disorder.

The MYB-NFIB gene fusion-a novel genetic link between adenoid cystic carcinoma and dermal cylindroma.

We have recently shown that the recurrent t(6;9)(q22 ∼ 23;p23 ∼ 24) translocation in adenoid cystic carcinoma (ACC) of the breast and head and neck results in a fusion of the two transcription factor genes MYB and NFIB. Here we demonstrate, for the first time, that benign sporadic, dermal cylindromas also express the MYB-NFIB gene fusion. RT-PCR and immunohistochemical analyses revealed that eight of 12 analysed tumours (67%) expressed MYB-NFIB fusion transcripts and/or stained positive for MYB protein. Nucleotide sequence analyses confirmed that the composition of the chimeric transcript variants identified was identical to that in ACC, suggesting a similar molecular mechanism of activation of MYB in cylindroma as in ACC. In contrast, no evidence for the presence of the MYB-NFIB fusion was found in other types of basaloid skin and salivary gland tumours, indicating that the fusion indeed has a restricted expression pattern. Our findings broaden the spectrum of neoplasms associated with MYB oncogene activation and reveal a novel genetic link between ACC and dermal cylindroma. These results, together with our previous observations, further strengthen the evidence for common molecular pathways of importance for the development of both benign and malignant breast, salivary and adnexal tumours.

[Molecular markers in salivary gland tumors: their use in diagnostic and prognostic workup]. / Molekulare Marker in Speicheldrüsentumoren: Einsatz zur Diagnose und prognostischen Bewertung.

The molecular genetic background of salivary gland neoplasms has not been characterized in detail to date. However, interesting target genes which could be used as prognostic and diagnostic molecular biomarkers have already been identified, e.g. CRTC1-MAML2 in mucoepidermoid carcinoma, or PLAG1 and HMGA2 in pleomorphic adenoma. In particular, CRTC1-MAML2 has shown strong diagnostic and prognostic potential in recent years. One of the major advantages of molecular tumor markers is that valid results are obtained on minute cell and/or tissue samples. Due to high-throughput techniques like comparative genome hybridization (CGH), micro- or gene profiling array detection of new marker genes can be expected in the future. This is also true for the most frequent malignant salivary gland tumors after the mucoepidermoid carcinoma, i.e. adenoid cystic carcinomas and acinic cell carcinomas.

Organization and chromosomal localization of the human platelet-derived endothelial cell growth factor gene.

Human platelet-derived endothelial cell growth factor (hPD-ECGF) is a novel angiogenic factor which stimulates endothelial cell growth in vitro and promotes angiogenesis in vivo. We report here the cloning and sequencing of the gene for hPD-ECGF and its flanking regions. This gene is composed of 10 exons dispersed over a 4.3-kb region. Its promoter lacks a TATA box and a CCAAT box, structures characteristic of eukaryotic promoters. Instead, six copies of potential Sp1-binding sites (GGGCGG or CCGCCC) were clustered just upstream of the transcription start sites. Southern blot analysis using genomic DNAs from several vertebrates suggested that the gene for PD-ECGF is conserved phylogenetically among vertebrates. The gene for hPD-ECGF was localized to chromosome 22 by analysis of a panel of human-rodent somatic cell hybrid lines.

Fusion of the EWS-related gene TAF2N to TEC in extraskeletal myxoid chondrosarcoma.

Extraskeletal myxoid chondrosarcomas (EMCs) are characterized by a recurrent t(9;22)(q22;q12) translocation, resulting in the fusion of the EWS gene in 22q12 and the TEC gene in 9q22. Here we report that a third member of the EWS, TLS/FUS gene family, TAF2N, can replace EWS as a fusion partner to TEC in EMC. Two tumors, one with a novel t(9;17)(q22;q11) variant translocation and one with an apparently normal karyotype, expressed TAF2N-TEC fusion transcripts. In both cases, the chimeric transcripts were shown to contain exon 6 of TAF2N fused to the entire coding region of TEC. This transcript is structurally and functionally very similar to the EWS-TEC fusions. The exchange of the EWS NH2-terminal part with the TAF2N NH2-terminal part in EMC further underscores the oncogenic potential of these protein domains as partners in fusion genes.

Fusion of the NH2-terminal domain of the basic helix-loop-helix protein TCF12 to TEC in extraskeletal myxoid chondrosarcoma with translocation t(9;15)(q22;q21).

Extraskeletal myxoid chondrosarcomas (EMCs) are characterized by recurrent t(9;22) or t(9;17) translocations resulting in fusions of the NH2-terminal transactivation domains of EWS or TAF2N to the entire TEC protein. We report here an EMC with a novel translocation t(9; 15)(q22;q21) and a third type of TEC-containing fusion gene. The chimeric transcript encodes a protein in which the first 108 amino acids of the NH2-terminus of the basic helix-loop-helix (bHLH) protein TCF12 is linked to the entire TEC protein. The translocation separates the NH2-terminal domain of TCF12 from the bHLH domain as well as from a potential leucine zipper domain located immediately downstream of the breakpoint. These results demonstrate that the NH2-terminal transactivation domains of EWS or TAF2N are not unique in their ability to convert the TEC protein into an oncogenically active fusion protein, and that they may be replaced by a domain from a bHLH protein that presumably endows the fusion protein with similar functions.

Expression of reciprocal hybrid transcripts of HMGIC and FHIT in a pleomorphic adenoma of the parotid gland.

The developmentally regulated HMGIC gene, which encodes an architectural transcription factor, has recently been linked to the pathogenesis of benign solid tumors with chromosome aberrations involving 12q13-15. Among these tumors are pleomorphic adenoma of the salivary glands, lipoma, uterine leiomyoma, hamartomas of the breast and lung, fibroadenoma of the breast, angiomyxoma, and endometrial polyps. For most tumor types, however, the translocation partners are variable. At present, no translocation partner genes of HMGIC are known for pleomorphic adenomas. Here, we report that in a primary pleomorphic adenoma of the parotid gland, the FHIT gene, which spans the chromosome 3p14.2 fragile site FRA3B, and is frequently disrupted in tumors, acts as a fusion partner of HMGIC. In addition to normal HMGIC and FHIT transcripts, an HMGIC/FHIT hybrid transcript as well as its reciprocal counterpart, FHIT/HMGIC, were found to be expressed by reverse transcription-PCR. The results establish the concurrent disruption of two tumor-associated genes in a benign tumor.

Escape from senescence in hybrid cell clones involves deletions of two regions located on human chromosome 1q.

Human normal cells have been shown to undergo a limited number of cell doublings, a phenomenon termed cellular senescence. Human chromosome 1 has been implicated in this process, and several lines of evidence indicate that there is a senescence-inducing gene or genes on human chromosome 1q. Our approach to analyze the senescence-inducing effect of chromosome 1 includes the use of somatic cell hybrid revertants. We show here that fusion of a hypoxanthine phosphoribosyl transferase-negative mouse cell line (A9) containing a human neo-tagged chromosome 1 with an immortal hamster cell line (10W-2) results in cell hybrids that senesce after a few population doublings. Rare revertants that had escaped senescence were obtained after one large fusion experiment. Thirty-five nonsenescent hybrids were obtained from a total of approximately 1 million hybrids, and 25 of these were subjected to further analysis. The presence of a single copy of human chromosome 1 in the revertant hybrids was confirmed by fluorescence in situ hybridization analysis using a chromosome 1-specific painting probe. No visible translocations or deletions of chromosome 1 were observed in any of the hybrids. Deletion mapping revealed that 11 (56%) of the hybrids analyzed had lost one or more markers on chromosome 1q. Two regions with deletions were detected, one of which has been shown to be implicated in the senescence-inducing effect exerted by chromosome 1 following monochromosome transfer (P. J. Vojta et al., manuscript submitted for publication). The present study suggests that two separate loci on human chromosome 1q may be of importance for the induction of senescence. Moreover, this set of nonsenescent revertants could be useful for future detailed analyses of the senescence-inducing loci.

Human glial fibrillary acidic protein: complementary DNA cloning, chromosome localization, and messenger RNA expression in human glioma cell lines of various phenotypes.

Glial fibrillary acidic protein (GFAP) is a constituent of intermediate filaments of glial cells of the astrocyte lineage. We cloned a human GFAP complementary DNA, deduced the amino acid sequence, and established the chromosomal location (17q21) of the GFAP gene by Southern blot hybridization of somatic cell hybrids and by in situ hybridization. The authenticity of the complementary DNA was proven by expressing it in glioma cells lacking endogenous GFAP; after microinjection of the complementary DNA, such cells became positive for staining with GFAP antibodies. The levels of fibronectin (FN) and GFAP mRNA of ten human glioblastoma cell lines, determined by Northern blot hybridization of RNA, were related to other phenotypic characteristics [cell morphology and expression of the genes encoding platelet-derived growth factor (PDGF) receptors]. A high expression of GFAP mRNA was found only in cells lacking fibronectin mRNA and protein. Glioma cells with a fibroblastic phenotype (bipolar, FN+/GFAP-) were found to express both types of PDGF receptors (alpha and beta). Relatively high levels of PDGF alpha-receptor mRNA, in the absence of beta-receptor expression, were found in cell lines that express GFAP and lack detectable levels of fibronectin mRNA. The findings are compatible with the idea that the genes encoding PDGF receptors in glioma cells are regulated in concert with other genes, the expression of which may reflect the developmental program of normal glia cell lineages.

Conserved mechanism of PLAG1 activation in salivary gland tumors with and without chromosome 8q12 abnormalities: identification of SII as a new fusion partner gene.

We have previously shown (K. Kas et al, Nat. Genet., 15: 170-174, 1997) that the developmentally regulated zinc finger gene pleomorphic adenoma gene 1 (PLAG1) is the target gene in 8q12 in pleomorphic adenomas of the salivary glands with t(3;8)(p21;q12) translocations. The t(3;8) results in promoter swapping between PLAG1 and the constitutively expressed gene for beta-catenin (CTNNB1), leading to activation of PLAG1 expression and reduced expression of CTNNB1. Here we have studied the expression of PLAG1 by Northern blot analysis in 47 primary benign and malignant human tumors with or without cytogenetic abnormalities of 8q12. Overexpression of PLAG1 was found in 23 tumors (49%). Thirteen of 17 pleomorphic adenomas with a normal karyotype and 5 of 10 with 12q13-15 abnormalities overexpressed PLAG1, which demonstrates that PLAG1 activation is a frequent event in adenomas irrespective of karyotype. In contrast, PLAG1 was overexpressed in only 2 of 11 malignant salivary gland tumors analyzed, which suggests that, at least in salivary gland tumors, PLAG1 activation preferentially occurs in benign tumors. PLAG1 over-expression was also found in three of nine mesenchymal tumors, i.e., in two uterine leiomyomas and one leiomyosarcoma. RNase protection, rapid amplification of 5'-cDNA ends (5'-RACE), and reverse transcription-PCR analyses of five adenomas with a normal karyotype revealed fusion transcripts in three tumors. Nucleotide sequence analysis of these showed that they contained fusions between PLAG1 and CTNNB1 (one case) or PLAG1 and a novel fusion partner gene, i.e., the gene encoding the transcription elongation factor SII (two cases). The fusions occurred in the 5' noncoding region of PLAG1, leading to exchange of regulatory control elements and, as a consequence, activation of PLAG1 gene expression. Because all of the cases had grossly normal karyotypes, the rearrangements must result from cryptic rearrangements. The results suggest that in addition to chromosomal translocations and cryptic rearrangements, PLAG1 may also be activated by mutations or indirect mechanisms. Our findings establish a conserved mechanism of PLAG1 activation in salivary gland tumors with and without 8q12 aberrations, which indicates that such activation is a frequent event in these tumors.

Structural alterations of the c-mos locus in benign pleomorphic adenomas with chromosome abnormalities of 8q12.

Recent mapping studies have assigned the human c-mos proto-oncogene to chromosome 8, bands q11-12. This region is frequently affected by chromosomal translocations in benign pleomorphic adenomas of the salivary glands. Using Southern blot analysis we report here that the c-mos gene and its flanking sequences are structurally altered in pleomorphic adenomas with chromosomal rearrangements of 8q12. Rearrangements were detected in two out of 23 tumors. Restriction fragment analysis indicated that the rearrangements were due to multiple, subtle mutations involving the c-mos open reading frame and its flanking sequences. There was no direct evidence of translocation of mos in any of the tumors. Control DNAs from the two patients showed a normal restriction pattern for all enzymes tested, indicating that the rearrangements are tumor specific. Collectively, our cytogenetic and molecular data suggest involvement of the c-mos gene in the pathogenesis of pleomorphic adenomas.

The recurrent translocation t(5;8)(p13;q12) in pleomorphic adenomas results in upregulation of PLAG1 gene expression under control of the LIFR promoter.

We have previously shown that the PLAG1 gene on chromosome 8q12 is consistently rearranged in pleomorphic adenomas of the salivary glands with t(3;8)(p21;q12) translocations. The t(3;8) results in promoter swapping between the PLAG1 gene, which encodes a novel zinc finger protein, and the constitutively expressed gene for beta-catenin (CTNNB1), a protein with roles in cell-cell adhesion and the WG/WNT signalling pathway. In order to assess the importance of other translocation partner genes of PLAG1, and their possible relationship to CTNNB1, we have characterized a second recurrent translocation, i.e. the t(5;8)(p13;q12). This translocation leads to ectopic expression of a chimeric transcript consisting of sequences from the ubiquitously expressed gene for the leukemia inhibitory factor receptor (LIFR) and PLAG1. As for the t(3;8), the fusions occurred in the 5'-noncoding regions of both genes, exchanging regulatory control elements while preserving the coding sequences. The results of the current as well as previous studies indicate that ectopic expression of PLAG1 under the control of promoters of distinct translocation partner genes is a general pathogenetic mechanism for pleomorphic adenomas with 8q12 aberrations.

Identification of NFIB as recurrent translocation partner gene of HMGIC in pleomorphic adenomas.

Approximately 12% of all pleomorphic adenomas of the salivary glands are characterized by chromosome aberrations involving the chromosome segment 12q13-15. Several chromosomes have been found as translocation partners of chromosome 12, and some of these recurrently. Recently, the HMGIC gene was identified as the target gene affected by the 12q13-15 aberrations. Here, we report the identification and characterization of a new translocation partner gene of HMGIC in pleomorphic adenomas. 3'-RACE analysis of a primary adenoma with an apparently normal karyotype revealed an HMGIC fusion transcript containing ectopic sequences derived from the human NFIB gene, previously mapped to chromosome band 9p24.1. The HMGIC NFIB fusion transcript was also confirmed by RT-PCR. Since the chromosome segment 9p12-24 is repeatedly involved as translocation partner of chromosome 12q13-15 in pleomorphic adenomas, we tested whether NFIB might be a recurrent partner of HMGIC. RT-PCR analysis of a second adenoma with an ins(9;12)(p23;q12q15) as the sole anomaly, revealed that also in this tumor an HMGIC/NFIB hybrid transcript was present. The reciprocal NFIB/HMGIC fusion transcript, however, could not be detected in any of these tumors. Nucleotide sequence analysis of the fusion transcripts indicated that the genetic aberration in both tumors resulted in the replacement of a carboxy-terminal segment of HMGIC by the last five amino acids of NFIB. In conclusion, our results reveal the recurrent involvement of the NFIB gene as translocation partner gene of HMGIC in pleomorphic adenomas.

The human islet amyloid polypeptide (IAPP) gene. Organization, chromosomal localization and functional identification of a promoter region.

We report the isolation and characterization of the human gene encoding islet amyloid polypeptide (IAPP). Previously characterized cDNA sequences correspond to three exons of which the first is noncoding. A functional promoter region was identified in the 5' flanking DNA; however, this was farther upstream than expected. Northern blot analysis of human insulinoma RNA revealed three IAPP mRNAs of sizes 1.2, 1.8 and 2.1 kb, in agreement with three polyadenylation signals present in the 3' end of the gene. In situ hybridization to metaphase chromosomes resulted in two distinct peaks on chromosome 12, at 12p12-p13 and 12q13-q14. Southern blot analysis of genomic DNA suggested a single IAPP locus but also indicated the presence of additional homologous sequences in human genomic DNA.

Interphase cytogenetic analysis of cultured pleomorphic adenomas with a normal karyotype.

A subgroup of pleomorphic adenomas of the salivary glands is characterized by apparently normal stemline karyotypes. In this investigation we have used FISH to study whether clonal translocations involving chromosomes 8 and 12 might have escaped detection with conventional cytogenetic analysis. Whole chromosome painting probes for chromosomes 8 and 12 were hybridized to interphase nuclei from 22 cytogenetically normal pleomorphic adenomas. In 20 of the 22 cases the majority of the nuclei (75-93%) showed two hybridizing chromosome 8 and chromosome 12 domains of equal size, indicating the presence of two intact copies of each of these chromosomes. Evidence of clonal translocations was only detected in two tumors. These findings show that in the vast majority of adenomas with a normal karyotype we can exclude the possibility of an abnormal non-dividing or very slow growing, and thus undetected, tumor cell population in vitro. Our data provides further evidence to support the existence of a subgroup of pleomorphic adenomas with a normal karyotype.

Fish analysis of supernumerary ring chromosome in dermatofibrosarcoma protuberans.

We describe the light- and electron microscopic, immunohistochemical, cell culture characteristics and cytogenetic findings of a case of dermatofibrosarcoma protuberans (DFSP). Cytogenetically, the lesion exhibited trisomy 8 and a supernumerary ring chromosome as the only clonal abnormalities found in about 35% of the cells analyzed. FISH-analysis of metaphase chromosomes revealed that the ring chromosome contained chromosome 17 sequences. Hybridization with a chromosome 17 centromere specific probe gave three signals in about 19% of the interphase nuclei suggesting that the ring also had a centromere derived from chromosome 17. These observations add to the evidence that supernumerary ring chromosomes, preferentially derived from chromosome 17, and trisomy 8 are non-random abnormalities in DFSP. Our findings also demonstrate the usefulness of FISH for identifying the origin of marker ring chromosomes.

Evidence of involvement of the PLAG1 gene in lipoblastomas.

Previous cytogenetic studies have demonstrated that the majority of lipoblastomas show rearrangements, in particular translocations and insertions, with breakpoints in 8q11-13. Here we present evidence for involvement of the developmentally regulated zink finger gene PLAG1 in these rearrangements. Northern blot and RT-PCR analyses revealed overexpression of PLAG1 in two lipoblastomas. Using immunohistochemistry, expression of the PLAG1 protein was also demonstrated in tissue sections from two lipoblastomas, one of which had a t(3;8)(q13.1;q12) translocation and the other a t(1;6)(q42;p22) translocation. Since no aberrant PLAG1 transcripts could be detected, it is likely that the gene may be activated by promoter swapping/substitution or alternatively by an as yet unknown mechanism. Our findings indicate that PLAG1 activation is a recurrent event in lipoblastomas and that PLAG1 is likely to be the target gene on chromosome 8 in these tumors.