High-resolution array CGH analysis of salivary gland tumors reveals fusion and amplification of the FGFR1 and PLAG1 genes in ring chromosomes.

We have previously identified a subgroup of pleomorphic salivary gland adenomas with ring chromosomes of uncertain derivation. Here, we have used spectral karyotyping (SKY), fluorescence in situ hybridization (FISH) and high-resolution oligonucleotide array-CGH to determine the origin and content of these rings and to identify genes disrupted as a result of ring formation. Of 16 tumors with rings, 11 were derived from chromosome 8, 3 from chromosome 5 and 1 each from chromosomes 1, 6 and 9. Array-CGH revealed that 10/11 r(8) consisted of amplification of a 19 Mb pericentromeric segment with recurrent breakpoints in FGFR1 in 8p12 and in PLAG1 in 8q12.1. Molecular analyses revealed that ring formation consistently generated novel FGFR1-PLAG1 gene fusions in which the 5'-part of FGFR1 is linked to the coding sequence of PLAG1. An alternative mechanism of PLAG1 activation was found in tumors with copy number gain of an intact PLAG1 gene. Rings derived from chromosomes 1, 5, 6 or 9 did not result in gene fusions, but rather resulted in losses indicative of the involvement of putative tumor suppressor genes on 8p, 5p, 5q and/or 6q. Our findings also reveal a novel mechanism by which FGFR1 contributes to oncogenesis and further illustrate the versatility of the FGFR1 and PLAG1 genes in tumorigenesis.

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.

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.

Cytogenetics of parametrial leiomyoma.

The cytogenetical findings from a parametrial leiomyoma are presented. The results, together with those of five previously presented cases, show obvious differences when compared to the chromosomal findings in uterine myomas. Ontogenic factors are proposed to be causative for the cytogenetical differences.

Impact of the in vitro technique used on the cytogenetic patterns in pleomorphic adenomas.

The cytogenetic observations in 97 new cases of pleomorphic adenomas are reported. They were all studied by in vitro technique using enzymatic pretreatment of the explanted material. The results, together with 26 previously reported cases studied by the same method (series II), were compared with a group of 130 adenomas (series I) investigated in cultures of only mechanically dispersed material. The results showed that in series II there was a substantial reduction of normal stemlines, an increase of cases with t(3;8)(p21;q12) and, in particular, of those with other 8q12 involvement. There was a similar frequency of cases with 12q13-15 aberrations in both series whereas, in series II, there was a moderate increase of the cases with "unique" abnormal stemlines. In comparison the German material, comprising 220 cases also studied by the enzymatic method, revealed a much higher frequency of cases with a normal stemline and a much lower frequency of adenomas with 8q12-involvement and of cases with unique stemlines. The results emphasize the need and importance of a meticulous documentation of all details of the in vitro technique used, for instance: explantation methods, composition of media, growth periods in vitro, and methods of harvest.

Cytogenetical observations in a cultured polymorphous low-grade adenocarcinoma originating from the minor salivary glands.

The cytogenetical findings in a cultured polymorphous low-grade adenocarcinoma (PLGA) of the minor salivary glands are reported. The deviations observed showed similarities to those found in the only hitherto studied case originating in the minor glands. Both these cases, however, showed a picture completely different from that in the two reported cases of parotid PLGA, constituting the malignant component in carcinomas ex pleomorphic adenoma. The most probably reasons are believed to be aetiological differences.

Cytogenetical observations in a mucoepidermoid carcinoma arising from heterotopic intranodal salivary gland tissue.

The cytogenetical observations in a mucoepidermoid carcinoma (MEG) arising from heterotopic intranodal salivary gland tissue are presented. The MEC was characterized by two reciprocal translocations, viz, t(1;7) (p36;q11) and t(11;19) (q14-21;p11). The present and the previous studies show that the last rearrangement or related deviations characterize about 40% of all MEG. The remaining cases form a subgroup distinguished by trisomies with or without concomitant structural deviations other than those affecting 11q14-22. At least MEC of the first subgroup show neither predilection for any specific anatomical site nor for any age group or particular sex. Inducing agent(s) and genotypic status of the tissue of origin seem to be the decisive factors for the chromosomal patterns in MEG.

Molecular analysis of uterine leiomyomas using the probes D7S13(7q) and HSPA2(14q).

Human uterine leiomyomas, mostly cytogenetically characterized, were analyzed by Southern blotting. The region 7q22.3-q31.2 was analyzed in 54 leiomyomas using the polymorphic probe D7S13. The region 14q22-24 was investigated in 25 leiomyomas with the probe HSPA2. No evidence of rearrangement or amplification of these DNA-regions was observed. Nor was any loss of polymorphism for the probe D7S13 seen. These, and earlier negative results, motivate further molecular investigation to understand the mechanisms reflected by recurrent chromosome aberrations in myomas.

Is pleomorphic adenoma of the salivary glands a tumor of congenital or very early origin?

The cytogenetical observations in a cultured parotid pleomorphic adenoma are presented. The patient had received treatment with X-rays for an infectious disorder in the same region about 50 years earlier. A polyclonal chromosomal pattern was disclosed with predominance of unique structural deviations. We have observed a similar chromosomal picture in a previously reported adenoma. This patient had also been treated with X-rays about 50 years earlier. The findings in these two cases suggest that many, perhaps all, pleomorphic adenomas are either congenital or arise very early in life. Their clinical manifestation at strongly varying time points in adulthood would then depend on accumulated mutational effects of unknown oncogenic agents.

Cytogenetical observations and hormone receptor expression in five extra-uterine leiomyomas.

The chromosomes were studied by an in vitro technique in four parametrial and one gastric leiomyomas. The findings in parametrial myomas (the present four cases and one published earlier) differed from the observations in uterine cases by the absence of normal stemlines and the absence of 1p,6p and 7q changes. As has been shown earlier for intraocular melanomas, the location within an organ or organ system could be the explanation for the chromosomal differences. The scanty data for leiomyomas in the digestive tract (the present case and two in the literature) suggest evolutionary patterns different from those in myomas in the genital tract. The reasons for this are unknown but differences in hormonal sensitivity, as reflected by estrogen and progesterone receptor expression, might at least be a contributory factory.

A study on the oncogenes gli, fos, jun and met in human uterine leiomyomas.

122 human uterine leiomyomas, representing the main chromosomal aberrations seen in these tumours, were analyzed by Southern blotting. Probes for the four oncogenes, gli, fos, jun, and met, all localized in or close to the chromosomal breakpoints in leiomyomas, were used. In only one single leiomyoma, with a 7p+q- marker, was there evidence of rearrangement of one of the oncogenes, namely met.

Cytogenetical and fish analysis on a salivary sebaceous lymphadenoma.

Chromosomal studies of a cultured parotid sebaceous lymphadenoma showed two abnormal cell groups, one with trisomy 9 (15% of the metaphases) and one with inconsistent structural deviations (10%). FISH analysis showed that the trisomic clone was actually larger (36%) than that revealed by chromosomal studies. The sebaceous lymphadenoma represents the fourth type of benign salivary gland tumor to show clonal chromosomal deviations.

Complex chromosomal changes in a primary squamous carcinoma of the parotid gland.

The chromosomal observations in a cultured primary epidermoid carcinoma of the parotid gland are reported. The tumour had a flat hyper-triploid mode with 7 recurrent wholly or partially identified marker types and 7-13 additional, mostly recurrent, markers, whose origin could not be clarified. There were also many recurring numerical deviations in most tumour cells. The picture was consistent with a neoplasma in an advanced stage of chromosomal progression. So far, 6q-markers with varying morphology are the only deviations found in most types of malignant salivary tumours and, in particular, in a high percentage of adenoid-cystic carcinomas. One possible explanation for these observations is the occurrence of one or more suppressor genes in 6q which may have relevance for malignant neoplasia in salivary gland tissues.

Human benign chondroblastoma with a pseudodiploid stemline characterized by a complex and balanced translocation.

The chromosomes of a human benign chondroblastoma of the jaw were studied by in vitro technique. Approximately one-third of the analyzed cells had a normal karyotype. The remaining two-thirds constituted an abnormal monoclonal population with a complex and balanced translocation. The observations are different from those in previously studied benign primary bone tumors. The breakpoint in 22q, however, is similar or very close to that observed in two extraskeletal myxoid chondrosarcomas earlier reported.

Karyotypic variability and evolutionary characteristics of a polymorphous low grade adenocarcinoma in the parotid gland.

The chromosomes of a polymorphous low-grade adenocarcinoma originating from a pleomorphic adenoma of the parotid gland were studied. Three successful preparations were performed. A minor fraction of cells showed normal karyotypes and some cells only inconsistent, usually numerical, deviations. The remaining cells constituted an abnormal monoclonal population with an unusual and very extensive karyotypic variability. The origin of most marker chromosomes could be wholly or partly clarified. Five different subclones could be distinguished on basis of different specific marker chromosomes. The characteristics of the marker sets suggested a closely interrelated derivation of the subclones. The results also provide insight as to the influence of random factors and/or differential growth rate on the chromosomal picture observed in in vitro systems. The present chromosomal observations showed no similarities either to the cytogenetical findings in the five previously reported salivary gland adenocarcinomas or to the deviations seen in the single studied case of carcinoma ex-pleomorphic adenoma.

Characterisation of a cell line (LCC-18) from a cultured human neuroendocrine-differentiated colonic carcinoma.

A cell line (LCC-18) from a neuroendocrine colonic tumour was established. The tumour cells retained their endocrine characteristics through more than 100 passages and showed positive immunocytochemistry for synaptophysin, vasoactive intestinal polypeptide (VIP) and glucagon. The culture medium also contained VIP and glucagon, which indicates that mechanisms for release of some of the active peptides were preserved. Transplantation of LCC-18 tumour cells into nude rats resulted in tumour formation with similar endocrine characteristics. The c-myc gene was amplified which might have been a prerequisite for establishment of the cell line. The chromosomes in LCC-18 were studied by G-banding and C-banding. The cell line had a distinctive mode in the hypotriploid region, at S = 61. The double minute (Dms) positive stemline karyotype showed numerical and structural aberrations more similar to findings in ordinary colonic adenocarcinomas than to observations in previously studied, pure intestinal neuroendocrine tumours. The Dms may be correlated with amplification of c-myc. LCC-18 may become valuable for studies of neuroendocrine differentiation, regulation of growth and production and release of hormones and for studies of drug effect.

Cytogenetics of multiple uterine leiomyomas, parametrial leiomyoma and disseminated peritoneal leiomyomatosis.

Using banding techniques the chromosomes were studied in 15 leiomyomas. The material comprised nine uterine myomas from one patient, one parametrial leiomyoma from a second patient and five tumors from a third patient with disseminated peritoneal leiomyomatosis. The results were considered together with pooled data from the literature. From this it could be concluded that: (1) each leiomyoma was the product of a separate clonal development; (2) different leiomyomas from the same patient sometimes showed an identical abnormal stemline (possibly because of etiological influences); (3) uterine and extra-uterine leiomyomas seemed to follow similar evolutionary pathways in their chromosomal progression.

Chromosomal patterns in human benign uterine leiomyomas.

Chromosomal observations by banding technique in 18 short-term cultured human uterine leiomyomas are reported. Half of the tumors had a primary or secondary abnormal stemline. They were usually characterized only by structural changes, in particular reciprocal translocations or insertions. Reviewing already published cases together with the new material confirmed that the aberrations in abnormal stemlines predominantly affected chromosomes 1, 2, 6, 7, 12, 14, and X. In these chromosomes the regions 1p36, 2p24, 6p12-21, 7q21-31, 12q13-15, 14q22-24, and the short arm of the X chromosome were preferentially affected. As in two other thoroughly studied human benign tumors, the pleomorphic adenoma and the meningioma, the very specific but sometimes complex chromosomal aberrations in leiomyomas could well be events of primary evolutionary importance. Likewise, in cases with a normal stemline, it is possible that comparable changes in the corresponding specific chromosomal regions have occurred at a submicroscopic level. Ascertaining this possibility, as well as the role of the aberrations with regard to the benign nature of the tumors, must be the focus of future analysis using molecular techniques.