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.

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.

POU5F1, encoding a key regulator of stem cell pluripotency, is fused to EWSR1 in hidradenoma of the skin and mucoepidermoid carcinoma of the salivary glands.

The EWSR1 gene is known to play a crucial role in the development of a number of different bone and soft tissue tumours, notably Ewing's sarcoma. POU5F1 is expressed during early development to maintain the totipotent status of embryonic stem and germ cells. In the present study, we report the fusion of EWSR1 and POU5F1 in two types of epithelial tumours: hidradenoma of the skin and mucoepidermoid carcinoma of the salivary glands. This finding not only broadens considerably the spectrum of neoplasms associated with EWSR1 fusion genes but also strengthens the evidence for shared pathogenetic mechanisms in the development of adnexal and salivary gland tumours. Reminiscent of the previously reported fusion genes involving EWSR1, the identified transcript is predicted to encode a chimeric protein consisting of the EWSR1 amino-terminal domain and the POU5F1 carboxy-terminal domain. We assessed the transcriptional activation potential of the chimera compared to the wild-type proteins, as well as activation of transcription through the oct/sox composite element known to bind POU5F1. Among other POU5F1 target genes, this element is present in the promoter of NANOG and in the distal enhancer of POU5F1 itself. Our results show that although the chimera is capable of significant transcriptional activation, it may in fact convey a negative regulatory effect on target genes.

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.

Cytogenetic and molecular genetic analyses of liposarcoma and its soft tissue simulators: recognition of new variants and differential diagnosis.

Liposarcoma is one of the most common sarcomas of adults. Its differential diagnosis and accurate subclassification are often problematic; the latter is also important with regard to appropriate treatment and prognosis. We studied a series of 23 liposarcomas that had unusual or previously undescribed features and 10 liposarcoma simulators and correlated the morphologic, cytogenetic, and molecular genetic findings. We found that use of cytogenetic-molecular genetic techniques aids in the distinction between myxoid-round cell liposarcoma and their simulators, chondroid lipoma, myxoid spindle cell-pleomorphic lipoma, cellular intramuscular myxoma, and myxofibrosarcoma. Poorly differentiated forms of round cell liposarcoma lacking morphologic evidence of lipogenesis can also be diagnosed using these techniques; however, the techniques do not aid in distinguishing low-grade myxoid from high-grade round cell liposarcomas. This study also shows that retroperitoneal liposarcomas with myxoid liposarcoma-like zones are part of the morphologic spectrum of well-differentiated-dedifferentiated liposarcoma rather than true myxoid liposarcomas. Perhaps most importantly, our results provide the first molecular genetic evidence that true mixed liposarcomas (mixed well-differentiated and myxoid liposarcoma) do indeed exist. They also unequivocally demonstrate the existence of small, round cell variants of pleomorphic liposarcoma that closely simulate myxoid-round cell liposarcoma.

A transplantable human carcinoid as model for somatostatin receptor-mediated and amine transporter-mediated radionuclide uptake.

A human midgut carcinoid tumor was successfully transplanted into nude mice and propagated for five consecutive generations (30 months) with well-preserved phenotype. Tumor cells in nude mice expressed identical neuroendocrine markers as the original tumor, including somatostatin receptors (somatostatin receptors 1 to 5) and vesicular monoamine transporters (VMAT1 and VMAT2). Because of the expression of somatostatin receptors and VMAT1 and VMAT2 the grafted tumors could be visualized scintigraphically using the somatostatin analogue 111In-octreotide and the catecholamine analogue 123I-metaiodobenzylguanidine. The biokinetics of the somatostatin analogue 111In-octreotide in the tumors was studied and showed a high retention 7 days after administration. Cell cultures were re-established from transplanted tumors. Immunocytochemical and ultrastructural studies confirmed the neuroendocrine differentiation. The human origin of transplanted tumor cells was confirmed by cytogenetic and fluorescence it situ hybridization analyses. Spontaneous secretion of serotonin and its metabolite, 5-hydroxyindole acetic acid, from tumor cells was demonstrated. The tumor cells increased their [Ca2+]i in response to beta-adrenoceptor stimulation (isoproterenol) and K+-depolarization. All somatostatin receptor subtypes could be demonstrated in cultured cells. This human transplantable carcinoid tumor, designated GOT1, grafted to nude mice, will give unique possibilities for studies of somatostatin receptor- and VMAT-mediated radionuclide uptake as well as for studies of secretory mechanisms.

Multicolor spectral karyotype analysis of a transplantable human ileal carcinoid.

In this report we present the results of a combined cytogenetic and multicolor spectral karyotype (SKY) analysis of a transplantable human ileal carcinoid (GOT1). By using SKY it was possible to identify the origin and organization of all clonal marker chromosomes and to identify cryptic translocations not detectable by conventional chromosome banding. The stemline karyotype of low passage GOT1 cells was interpreted as 43,XX, der(1)del(1)(?), inv(2)(p25q13), del(3)(p21), del(5)(q13q31), del(6)(q13), -9, -13, -15, del(16) (q22). Analysis of the GOT1 cells after about 2.5 years of propagation in nude mice allowed us to follow the in vivo progression of this tumor. Relatively few additional rearrangements had occurred during this period, indicating that the GOT1 cells are genetically stable. Most of the abnormalities detected result in loss of whole or parts of chromosomes, suggesting that loss of multiple chromosomal regions, presumably containing tumor suppressor genes, might be important genetic events in ileal carcinoids.

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.

Expression and mutation patterns of p53 in benign and malignant salivary gland tumors.

The expression and mutation patterns of p53 were studied in a series of 68 benign pleomorphic adenomas and 237 malignant salivary gland tumors. p53 overexpression (nuclear staining exceeding 10%) was detected in 20% of the malignant salivary gland tumors, with the highest prevalence observed in polymorphous low grade adenocarcinoma, squamous cell carcinoma, and carcinoma ex pleomorphic adenoma and the lowest in adenoid cystic carcinoma and acinic cell carcinoma. In contrast, none of the 68 benign pleomorphic adenomas had nuclear staining exceeding 10%. SSCP and nucleotide sequence analysis of exons 4 to 9 of p53 in 19 malignant tumors revealed 9 mutations in 7 tumors. Our findings indicate that p53 may be a useful marker to help discriminate between benign and malignant salivary gland tumors.

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.

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.

Large cell, epithelioid, telangiectatic osteoblastoma: a unique pseudosarcomatous variant of osteoblastoma.

A previously undescribed large-cell, epithelioid, and aneurysmal variant of osteoblastoma with minimal osteoid-production--simulating telangiectatic osteosarcoma, epithelioid angiosarcoma, and metastatic carcinoma is reported. The tumor occurred in the mandible of a 14-year-old girl. The light microscopic, immunohistochemical, ultrastructural, cell proliferation, and DNA-ploidy studies, as well as the 7-year disease-free follow-up period all indicate a benign osteoblastic tumor. Cytogenetically, the tumor had a pseudodiploid karyotype, distinguished by a complex t(1;5;17;22) and a terminal 1q deletion. Recognition of this unique, pseudomalignant variant of osteoblastoma is important to avoid an erroneous diagnosis of malignancy.

del(6)(q12q15) as the sole cytogenetic anomaly in a case of solitary infantile myofibromatosis.

We describe a case of light microscopically typical solitary, infantile myofibromatosis in a 6-month old boy. The myofibroblastic differentiation of the tumor was supported by immunohistochemical and ultrastructural analyses. Cytogenetic and FISH analyses revealed a pseudodiploid karyotype with an interstitial deletion of the long arm of one chromosome 6, del(6)(q12q15), as the sole anomaly. The results demonstrate the usefulness of cytogenetics and FISH in distinguishing this type of lesion from infantile fibrosarcoma. To the best of our knowledge this is the first cytogenetic analysis of solitary infantile myofibromatosis.

Chromosomal localization of three human genes encoding bone morphogenetic protein receptors.

Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily that play a pivotal role in bone formation during embryogenesis and fracture repair. BMP signaling occurs via hetero-oligomeric serine/threonine kinase complexes of BMP type I (BMPR-IA or BMPR-IB) and type II receptors (BMPR-II). BMPR-IA and IB are closely related receptors, with sequence differences conserved between different species, suggesting that they serve distinct functions. Here we report the cDNA cloning of human BMPR1B and the chromosomal localization of all three BMPR genes. Using somatic cell hybrid and FISH analyses, the BMPR1A, BMPR1B, and BMPR2 genes were assigned to 10q23, 4q22-24, and 2q33-34, respectively. A processed BMPR1A pseudogene was mapped to 6q23.

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.

Molecular analyses of the 15q and 18q SMAD genes in pancreatic cancer.

SMAD4 (DPC4) is part of the TGFB signaling pathway and is frequently inactivated in pancreatic carcinomas. TGFB signals from the membrane to the nucleus via SMAD proteins. TGFB receptor activation results in SMAD2 and SMAD3 phosphorylation, which then form heteromeric complexes with SMAD4. Inhibitory SMADs, SMAD6 and SMAD7, can prevent TGFB signaling by interacting either with the receptor or with SMAD2 and SMAD3. The encoding sequences for these proteins are organized in two gene clusters, one at 18q21 (SMAD2, SMAD4, and SMAD7) and the other at 15q21-22 (SMAD3 and SMAD6). Losses of 15q and 18q material are frequent in pancreatic carcinomas, and in order to map the extent of 15q and 18q deletions and to investigate further the involvement of SMAD4 and the possible function of SMAD2 and SMAD3 as tumor suppressor genes in pancreatic carcinoma, we performed loss of heterozygosity studies as well as mutation and expression analyses of SMAD4, SMAD2, and SMAD3 in 13 low-passage cell lines from 12 pancreatic carcinoma patients. To investigate possible amplifications of SMAD6 and SMAD7, the genomic organization and the expression levels of these genes were analyzed. One tumor with homozygous loss of SMAD4 was detected, and mutations of this gene were found in four of the 12 carcinomas; no SMAD2 or SMAD3 inactivating genomic alterations were found. In none of the cases was transcriptional silencing seen. No genomic amplifications, mutations, or increased expression of SMAD6 and SMAD7 were detected. These results suggest that functional abrogation of SMAD2 or SMAD3 and increased expression of SMAD6 or SMAD7 are infrequent in pancreatic carcinomas and further stress the particular importance of SMAD4 inactivation in pancreatic carcinogenesis.

Fluorescence in situ hybridization mapping of breakpoints in pleomorphic adenomas with 8q12-13 abnormalities identifies a subgroup of tumors without PLAG1 involvement.

Recently, we identified the PLAG1 gene as the target gene in pleomorphic adenomas with chromosome abnormalities involving 8q12. The majority of breakpoints were shown to reside within the 5' noncoding region of the gene. We now report three pleomorphic adenomas with breakpoints located distal to PLAG1 in band 8q13. These tumors had the following chromosome 8 abnormalities: ins(8;12)(q12-13;q14q15), t(8;12)(q13;q15), and t(6;8)(p21.3-22;q13). Fluorescence in situ hybridization mapping of the chromosome 8 breakpoints revealed a yeast artificial chromosome clone spanning the breakpoints in two tumors. In none of the cases was PLAG1 activated and/or disrupted. Three candidate genes, N8, HMGIC, and HMGIY, were analyzed for rearrangements and/or abnormal expression by using reverse transcriptase-polymerase chain reaction, rapid amplification of 3' cDNA ends, and Northern blot analyses.

A child with a t(11;19)(q14-21;p12) in a pulmonary mucoepidermoid carcinoma.

We report on a mucoepidermoid carcinoma (MEC) of the lung in a 6-year-old girl with a t(11;19)(q14-21;p12) as the sole karyotypic abnormality. An apparently identical t(11;19) has been reported previously in a MEC originating from the major and minor salivary glands. Our findings indicate that the t(11;19) is intimately associated with the mucoepidermoid phenotype and may be used as a diagnostic marker for this tumour type.