Basaloid lesions of oral squamous epithelial cells and their association with HPV infection and P16 expression.

UNLABELLED: Basaloid squamous cell carcinoma is a variant of squamous cell carcinoma, preferentially arising in the head and neck region. Current reports point to an association of basaloid squamous cell carcinoma (BSCC) and human papilloma virus (HPV) infection. This virus is supposed to be an aetiological factor in this entity. The aim of this study was to analyse the HPV infection status in different entities of oral neoplasms or dysplasias with basaloid differentiation of epithelial cells. MATERIALS AND METHODS: The study comprised data from 34 oral lesions of squamous epithelial origin: 17 BSCCs, 10 papillomas and 7 hyperplasias/dysplasias of oral epithelia. HPV DNA was detected by means of a hybrid capture technique. HPV types were identified by direct sequencing. Immunohistochemical investigation of the specimens with anti-p16 antibody was performed in order to elucidate the putative role of p16 as a surrogate marker of HPV infection. RESULTS: The rate of HPV-infected BSCC was extraordinarily high. About two thirds of the cases (61%, 11/17) were infected with HPV high-risk types, predominantly with HPV genotype 16 (>90%, 10/11). The infection status differed significantly between BSCC and other oral lesions in terms of frequency of HPV infection and HPV genotypes. p16 expression proved not to be a suitable surrogate marker of HPV high-risk infection in oral lesions, in particular in BSCC. This was an essential difference of this collective compared to genital carcinomas with HPV high-risk infections. CONCLUSION: This study revealed a high association between BSCC and HPV type 16. This close phenotype-genotype correlation could be of diagnostic value. Type-specific analysis of HPV infection in head and neck cancer may be important in the differential diagnosis of malignancies in the head and neck region with a basaloid growth pattern. However, the investigation is technically demanding, including hybridisation and sequencing techniques. A simplified test for HPV in BSCC of the oral cavity using the immunohistochemical proof of p16 expression as a surrogate marker is non-effective.

Expression of proteases in giant cell lesions of the jaws, tendon sheath and salivary glands.

INTRODUCTION: Peripheral giant cell granuloma (GC) of the jaw is a tumour-like lesion, situated on the gingiva. The aim of this study was to: (a) better define the cellular compartments of the lesion and (b) compare the protease expression-profile in GC lesions of the jaws to GC lesions of other sites. MATERIALS AND METHODS: This study comprised 54 GC lesions (jaws: 30, tendon sheaths: 22, salivary glands: 2). A microarray technique was applied to the study of osteoclast-specific or osteoclast-like features of different sites (CD68, CD51, RANK, M-CSF). Proteases were immunohistochemically identified [cathepsin K, L, S and matrix metalloproteinase 9 (MMP9)]. RESULTS: The GC of all lesions were immunoreactive for CD68 and CD51. Factors indicating the differentiation and activation of osteoclasts were detected in all lesions (RANK, M-CSF, cathepsin K, MMP9). The expression profile of M-CSF in GC and stroma cells was of a medium grade in cases with no apparent destruction of bone, whereas RANK was expressed only weakly in mono- or multinuclear CD68-positive cells. CONCLUSION: The results of this study reveal an identical cellular composition for all lesions irrespective of site. GC of lesions at all sites contain the same osteolytic proteases and express cytokines that are effective in bone metabolism. The reason for the absence of osteolysis in some 'epulis' cases may be due to the topography of the lesion. Furthermore, the reduced number of binding sites, revealed by the low expression profile of RANK, may possibly be responsible for an absence of or only superficial osteolysis in these cases, despite evidence of M-CSF.

A link between the expression of the stem cell marker HMGA2, grading, and the fusion CRTC1-MAML2 in mucoepidermoid carcinoma.

Recently, the concept of cancer stem cells and their expression of embryonic stem cell markers has gained considerable experimental support. In this study, we examined the expression of one such marker, the high-mobility group AT-hook 2 gene (HMGA2) mRNA, in 53 formalin-fixed, paraffin-embedded mucoepidermoid carcinomas (MEC) and four normal parotid tissues using quantitative real-time RT-PCR (qPCR). MECs are often characterized by the fusion gene CRTC1-MAML2, the detection of which is an important tool for the diagnosis and prognosis of MEC. For detection of the CRTC1-MAML2 fusion transcript, we performed RT-PCR. The mean expression level of HMGA2 was higher in fusion negative (302.8 +/- 124.4; n = 14) than in positive tumors (67.3 +/- 13.1; n = 39). Furthermore, the fusion-negative tumors were often high-grade tumors and the HMGA2 expression level rose with the tumor grade (low: 43.7 +/- 11.0, intermediate: 126.2 +/- 28.3, and high: 271.2 +/- 126.5). A significant difference was found in the HMGA2 expression levels between the different grading groups (one-way ANOVA, P = 0.04) and among the fusion-negative and -positive tumors (t-test, P = 0.05), indicating that the expression level of HMGA2 was closely linked to grading, the presence/absence of the CRTC1-MAML2 fusion, and the tumor behavior of MECs. These findings offer further evidence for the theory that the MEC group comprises two subgroups: one group with the CRTC1-MAML2 fusion, which is a group with a moderate aggressiveness and prognosis, and the other group lacking that fusion corresponding to an increased stemness, and thus, higher aggressiveness and worse prognosis.

Copy number gain at 8q12.1-q22.1 is associated with a malignant tumor phenotype in salivary gland myoepitheliomas.

Salivary gland myoepithelial tumors are relatively uncommon tumors with an unpredictable clinical course. More knowledge about their genetic profiles is necessary to identify novel predictors of disease. In this study, we subjected 27 primary tumors (15 myoepitheliomas and 12 myoepithelial carcinomas) to genome-wide microarray-based comparative genomic hybridization (array CGH). We set out to delineate known chromosomal aberrations in more detail and to unravel chromosomal differences between benign myoepitheliomas and myoepithelial carcinomas. Patterns of DNA copy number aberrations were analyzed by unsupervised hierarchical cluster analysis. Both benign and malignant tumors revealed a limited amount of chromosomal alterations (median of 5 and 7.5, respectively). In both tumor groups, high frequency gains (> or =20%) were found mainly at loci of growth factors and growth factor receptors (e.g., PDGF, FGF(R)s, and EGFR). In myoepitheliomas, high frequency losses (> or =20%) were detected at regions of proto-cadherins. Cluster analysis of the array CGH data identified three clusters. Differential copy numbers on chromosome arm 8q and chromosome 17 set the clusters apart. Cluster 1 contained a mixture of the two phenotypes (n = 10), cluster 2 included mostly benign tumors (n = 10), and cluster 3 only contained carcinomas (n = 7). Supervised analysis between malignant and benign tumors revealed a 36 Mbp-region at 8q being more frequently gained in malignant tumors (P = 0.007, FDR = 0.05). This is the first study investigating genomic differences between benign and malignant myoepithelial tumors of the salivary glands at a genomic level. Both unsupervised and supervised analysis of the genomic profiles revealed chromosome arm 8q to be involved in the malignant phenotype of salivary gland myoepitheliomas.

A closer look at Warthin tumors and the t(11;19).

The translocation t(11;19)(q21;p13) has been described in mucoepidermoid carcinoma (MEC) and rarely in Warthin tumors (WT), both tumors of the salivary gland. The translocation creates a fusion gene in which exon 1 of CRTC1 is linked to exons 2-5 of MAML2. To verify the translocation in WT, we performed nested reverse transcriptase-polymerase chain reaction using RNA from 48 WTs. This revealed the t(11;19)(q21;p13) translocation and expression of the chimeric gene in two metaplastic WT samples, but in none of the remaining ordinary 46 WTs. On review, the two positive cases were classified as tumors highly suspect for MEC. Indeed, our experience and published observations of the t(11;19)(q21;p13) translocation in WT reveal that only a small subset of WTs are positive, and that these tumors are often classified as infarcted or metaplastic WT, known to overlap considerably with MEC on purely morphological grounds. We therefore conclude that the presence of the t(11;19)(q21;p13) rearrangement favors a diagnosis of MEC.

A new type of MAML2 fusion in mucoepidermoid carcinoma.

The present study reports for the first time a CRTC3-MAML2 fusion gene in a mucoepidermoid carcinoma, as determined by RT-PCR and sequencing. We screened a total of 67 formalin-fixed, paraffin-embedded mucoepidermoid carcinomas for the presence of chimeric genes. In one of these samples, a CRTC3-MAML2 fusion gene was detected. Thus, this report demonstrates the existence of a fusion of MAML2 with CREB regulated transcriptional coactivator CRTC3 additional to the already known fusion of MAML2 and CRTC1. Both gene fusions seem to result in an identical tumor phenotype and the fusion genes CRTC1-MAML2 and CRTC3-MAML2 may play a similar role in the development of mucoepidermoid carcinomas.

Frequency of chromosomal aneuploidies and deletions of the RB and TP53 genes in MALT lymphomas harboring the t(14;18)(q32;q21).

The t(14;18)(q32;q21) involving the MALT1/MLT and IGH genes has been identified recently as a recurrent abnormality in mucosa-associated lymphoid tissue (MALT) lymphomas. The frequency of secondary chromosomal aberrations in MALT lymphomas harboring the t(14;18) is largely unknown. We therefore analyzed six t(14;18)-positive MALT lymphomas (five parotid, one conjunctiva) by interphase fluorescence in situ hybridization for aneuploidies of chromosomes 3, 7, 12, 18, and X, gains or disruption of the CMYC/8q24 and BCL6/3q27 genes, as well as deletions of the retinoblastoma and TP53 tumor suppressor genes. Except for one MALT lymphoma of the parotid with trisomy 3, neither aneuploidies nor deletions were detected in any of our cases.

Expression of the high-mobility group protein HMGI(Y) in gestational trophoblastic diseases.

The high-mobility group protein HMGI(Y) is a member of a family of nonhistone chromosomal proteins, which have been implicated in the regulation of inducible gene transcription, integration of retroviruses into chromosomes, and induction of neoplastic transformation and metastatic progression in cancer cells. The human trophoblast is a tissue that shares proliferation capacity and invasiveness with neoplastic tissues, but in which these processes are tightly regulated. Recently we could show that HMGI(Y) is expressed in the normal human placenta, where it is localized in the nuclei of villous cytotrophoblast, in the anchoring villi at the implantation site and in extravillous (intermediate) trophoblast invading the maternal decidua. In contrast, the majority of the nuclei of the villous syncytiotrophoblast, a terminally differentiated tissue, was negative. The purpose of this study was to investigate the expression pattern of HMGI(Y) in gestational trophoblastic diseases (GTD), which has not been studied so far. To analyze the expression of HMGI(Y), we performed immunohistochemistry on a total of 29 cases of GTD, including 21 hydatidiform moles and 8 choriocarcinomas. Hydatidiform moles showed a positivity for HMGI(Y) in villous cytotrophoblast and in areas of the trophoblast proliferations on the villous surface; villous syncytiotrophoblast was negative. The choriocarcinomas showed strong immunoreactivity in all cases. The expression pattern of HMGI(Y) in gestational trophoblastic diseases indicates that it might play a role in the pathogenesis of GTD and might be potentially useful as an additional diagnostic marker for such lesions.

[Salivary duct carcinomas comprise phenotypically and genotypically diverse high grade neoplasms]. / Speichelgangkarzinome--ein Spektrum phänotypisch und genotypisch heterogener Tumoren.

Salivary duct carcinomas (SDC) are high grade neoplasms morphologically reminiscent of breast ductal carcinomas. Whereas the latter are well characterized, the body of immunophenotypic and cytogenetic data on SDC is limited. We studied 23 SDC by conventional histology, immunohistology, in situ hybridization, and comparative genomic hybridization (CGH). Data were subjected to biomathematical analysis in comparison to previously characterized breast ductal carcinomas in situ and invasive ductal carcinoma cases. Most SDC stained for cytokeratins (Ck) Ck 8/18 (77 %) or Ck 5/6 (30 %), 30 % of cases expressed the androgen receptor (AR), 14 cases (63 %) expressed c-erbB2, and one case stained for prostate specific antigen. Except for two cases, Ck 8/18 and Ck 5/6 were not coexpressed. Ck 8/18 expression positively correlated with presence of c-erbB2 and AR. At variance, Ck 5/6 correlated positively with p63 and inversely with both AR and c-erbB2 expression. Ck 5/6 and p 63 co-expression was also found in a distinct population of ductal epithelial cells of normal salivary glands. CGH analysis of SDC revealed increasing numbers of alterations in correlation with advanced diseases, but no recurrent alterations. Cluster analysis of phenotypic and genotypic markers assigned both salivary and breast carcinomas to numerous clusters independent of the primary tumour site. Although undistinguishable by conventional histology, SDC are heterogeneous, comprising at least two immunophenotypically distinct subgroups of neoplasms. Cluster analysis suggests several distinct patterns of gene expression common to both primary sites explaining morphologic parallels between SDC and high grade breast cancer.

Neutron therapy, prognostic factors and dedifferentiation of adenoid cystic carcinomas (ACC) of salivary glands.

PURPOSE: Analysis of the efficacy of fast neutron radiotherapy in the treatment of adenoid cystic carcinomas (ACC) of the salivary glands, identification of prognostic variables and dedifferentiation after radiotherapy. PATIENTS AND METHODS: Histological slides of primary and recurrent lesions of 71 patients were reviewed to confirm the diagnosis and to analyse subtypes. Median follow-up was 52 months. Local control rate and overall survival were analysed in multivariate analysis. Complications are also described. RESULTS: Primary vs. recurrent therapy (p=0.001), margin-status (p=0.01) and subtype (p=0.019) influenced overall survival. Primary vs. recurrent therapy (p=0.001), margin-status (p=0.018) and T-stage (p=0.043) influenced local control rate. Dedifferentiation was seen in only 1/17 cases. CONCLUSION: The calculated prognostic factors illustrate the importance of a radical primary therapy. Histological subtype is a significant additional factor for overall survival and, in case of dedifferentiation, it is a strong predictor of a detrimental outcome.

Angiogenetic signaling through hypoxia: HMGB1: an angiogenetic switch molecule.

The initiation of angiogenesis, called the angiogenetic switch, is a crucial early step in tumor progression and propagation, ensuring an adequate oxygen supply. The rapid growth of tumors is accompanied by a reduced microvessel density, resulting in chronic hypoxia that often leads to necrotic areas within the tumor. These hypoxic and necrotic regions exhibit increased expression of angiogenetic growth factors, eg, vascular endothelial growth factor, and may also attract macrophages, which are known to produce a number of potent angiogenetic cytokines and growth factors. A group of molecules that may act as mediators of angiogenesis are the so-called high-mobility group proteins. Recent studies showed that HMGB1, known as an architectural chromatin-binding protein, can be extracellularly released by passive diffusion from necrotic cells and activated macrophages. To examine the angiogenetic effects of HMGB1 on endothelial cells an in vitro spheroid model was used. The results of the endothelial-sprouting assay clearly show that exogenous HMGB1 induced endothelial cell migration and sprouting in vitro in a dose-dependent manner. Thus, this is the first report showing strong evidence for HMGB1-induced sprouting of endothelial cells.

Expression of the high-mobility group protein HMGI(Y) in human trophoblast: potential role in trophoblast invasion of maternal tissue.

The high-mobility group protein HMGI(Y) is a member of a family of non-histone chromosomal proteins, which have been implicated in the regulation of inducible gene transcription, integration of retroviruses into chromosomes and induction of neoplastic transformation and metastatic progression in cancer cells. The human trophoblast is a tissue that shares proliferation capacity and invasiveness with neoplastic tissues, but in which these processes are tightly regulated. In the present study, we analyzed the expression of HMGI(Y) in the human placenta using immunohistochemistry. We found expression of HMGI(Y), with nuclear localization, in the villous cytotrophoblast (vCT), which is a highly proliferative cell type. In contrast, the majority of the nuclei of the villous syncytiotrophoblast, a terminally differentiated tissue, was negative. Interestingly, expression of HMGI(Y) was strongest in anchoring villi at the implantation site and in extravillous (intermediate) trophoblast (EVT) invading the maternal decidua. As vCT cells differentiate to become EVT, the HMGI(Y) protein appears to switch from a nuclear to a cytoplasmic localization. Expression of HMGI(Y) in isolated trophoblast populations in primary cell culture was also confirmed using Western-blot analysis. This study shows for the first time expression and localization of HMGI(Y) in the subpopulations of placental tissue.

Qualitative and quantitative observations of bone tissue reactions to anodised implants.

Research projects focusing on biomaterials related factors; the bulk implant material, the macro-design of the implant and the microsurface roughness are routinely being conducted at our laboratories. In this study, we have investigated the bone tissue reactions to turned commercially pure (c.p.) titanium implants with various thicknesses of the oxide films after 6 weeks of insertion in rabbit bone. The control c.p. titanium implants had an oxide thickness of 17-200 nm while the test implants revealed an oxide thickness between 600 and 1000 nm. Routine histological investigations of the tissue reactions around the implants and enzyme histochemical detections of alkaline and acid phosphatase activities demonstrated similar findings around both the control and test implants. In general, the histomorphometrical parameters (bone to implant contact and newly formed bone) revealed significant quantitative differences between the control and test implants. The test implants demonstrated a greater bone response histomorphometrically than control implants and the osteoconductivity was more pronounced around the test implant surfaces. The parameters that differed between the implant surfaces, i.e. the oxide thickness, the pore size distribution, the porosity and the crystallinity of the surface oxides may represent factors that have an influence on the histomorphometrical results indicated by a stronger bone tissue response to the test implant surfaces, with an oxide thickness of more than 600 nm.