Effects of HIFU induced cavitation on flooded lung parenchyma.

BACKGROUND: High intensity focused ultrasound (HIFU) has gained clinical interest as a non-invasive local tumour therapy in many organs. In addition, it has been shown that lung cancer can be targeted by HIFU using One-Lung Flooding (OLF). OLF generates a gas free saline-lung compound in one lung wing and therefore acoustic access to central lung tumours. It can be assumed that lung parenchyma is exposed to ultrasound intensities in the pre-focal path and in cases of misguiding. If so, cavitation might be induced in the saline fraction of flooded lung and cause tissue damage. Therefore this study was aimed to determine the thresholds of HIFU induced cavitation and tissue erosion in flooded lung. METHODS: Resected human lung lobes were flooded ex-vivo. HIFU (1,1 MHz) was targeted under sonographic guidance into flooded lung parenchyma. Cavitation events were counted using subharmonic passive cavitation detection (PCD). B-Mode imaging was used to detect cavitation and erosion sonographically. Tissue samples out of the focal zone were analysed histologically. RESULTS: In flooded lung, a PCD and a sonographic cavitation detection threshold of 625 Wcm- 2(pr = 4, 3 MPa) and 3.600 Wcm- 2(pr = 8, 3 MPa) was found. Cavitation in flooded lung appears as blurred hyperechoic focal region, which enhances echogenity with insonation time. Lung parenchyma erosion was detected at intensities above 7.200 Wcm- 2(pr = 10, 9 MPa). CONCLUSIONS: Cavitation occurs in flooded lung parenchyma, which can be detected passively and by B-Mode imaging. Focal intensities required for lung tumour ablation are below levels where erosive events occur. Therefore focal cavitation events can be monitored and potential risk from tissue erosion in flooded lung avoided.

Immunohistochemical and molecular characterizations in urothelial carcinoma of bladder in patients less than 45 years.

Bladder tumours in early-onset patients are rare and seem to exhibit unique clinicopathological features. Only few studies have investigated somatic alterations in this specific age of onset group and evidence is accumulating of a distinct molecular behaviour of early-onset bladder tumours. We collected the largest cohort of early-onset tumours of patients 45 years old or younger and aimed to test genomic alterations typically found in bladder cancer. Tumours of 118 early-onset patients were compared with a consecutive group of 113 cases. Immunohistochemistry of TP53, CK20 and Ki-67 was carried out. Molecular analysis was conducted to test for loss of heterozygosity of chromosome 9 and 17, as well as TP53 and FGFR3 mutations. Fisher´s exact and chi-squared test were appropriately used. No differences in grade/stage characteristics were observed. Overexpressed TP53 was differentially distributed between the two groups. TP53 nuclear accumulation was significantly more frequent in early-onset papillomas, PUNLMPs and pTa low-grade tumours compared to the consecutive cohort (p=0.005). Moreover, chromosome 9 deletions (29.5% vs. 44.6%) and FGFR3 mutations (34.5% vs. 63.7%) were less often detected in early-onset patients (p=0.05 and p<0.0001). By comparing the largest cohort of early-onset bladder cancer patients with an unselected group, we demonstrated that the typical molecular features are not independent of age at diagnosis. Our study supports the hypothesis of a distinct biological behaviour in early-onset tumours.

Flooded Lung Generates a Suitable Acoustic Pathway for Transthoracic Application of High Intensity Focused Ultrasound in Liver.

Background: In recent years, high intensity focused ultrasound (HIFU) has gained increasing clinical interest as a non-invasive method for local therapy of liver malignancies. HIFU treatment of tumours and metastases in the liver dome is limited due to the adjacent ultrasound blocking lung. One-lung flooding (OLF) enables complete sonography of lung and adjoining organs including liver. HIFU liver ablation passing through the flooded lung could enable a direct intercostal beam path and thus improve dose deposition in liver. In this study, we evaluate the feasibility of an ultrasound guided transthoracic, transpulmonary HIFU ablation of liver using OLF. Methods: After right-side lung flooding, ultrasound guided HIFU was applied transthoracic- transpulmonary into liver to create thermal lesions in three pigs. The HIFU beam was targeted five times into liver, two times at the liver surface and three times deeper into the tissue. During autopsy examinations of lung, diaphragm and liver located in the HIFU path were performed. The focal liver lesions and lung tissue out of the beam path were examined histologically. Results: Fifteen thermal liver lesions were generated by transpulmonary HIFU sonication in all targeted regions. The lesions appeared well-demarcated in grey color with a cigar-shaped configuration. The mean length and width of the superficial and deeper lesions were 15.8 mm (range: 13-18 mm) and 5.8 mm (range: 5-7 mm), and 10.9 mm (range: 9-13 mm) and 3.3 mm (range: 2-5 mm), respectively. Histopathological, all liver lesions revealed a homogeneous thermal necrosis lacking vitality. There were no signs of damage of the overlying diaphragm and lung tissue. Conclusions: Flooded lung is a suitable pathway for applying HIFU to the liver, thus enabling a transthoracic, transpulmonary approach. The enlarged acoustic window could enhance the ablation speed for targets in the hepatic dome.

Recurrent mutations within the amino-terminal region of ß-catenin are probable key molecular driver events in sinonasal hemangiopericytoma.

Sinonasal hemangiopericytoma (SN-HPC) is an uncommon, site-specific, low-grade mesenchymal neoplasm of probable perivascular myoid cell origin. In contrast to solitary fibrous tumors of soft tissue and sinonasal tract origin, SN-HPCs were recently shown to lack recurrent NAB2-STAT6 fusion variants. Other molecular alterations known to occur in some of soft tissue perivascular myoid cell neoplasms were also absent in SN-HPC; thus, the molecular pathogenesis of SN-HPCs remained unknown. Guided by whole-genome sequencing combined with RNA sequencing of an index case, we analyzed a total of six SN-HPCs for mutations within the amino-terminal region of the gene CTNNB1 (cadherin-associated protein), ß 1, 88 kDa, encoding ß-catenin. All six cases showed missense mutations, with amino acid substitutions clustering at positions 33 to 45, corresponding to the recognition site of the ß-catenin destruction complex. Similar CTNNB1 mutations have been described in a variety of epithelial and mesenchymal neoplasms. These mutations prevent ß-catenin phosphorylation and proteasomal degradation but promote its nuclear accumulation and subsequent increased transcription of Wingless-related integration site target genes. Consistent with these molecular findings, ß-catenin IHC showed consistent diffuse and strong nuclear staining of the tumor cells in all six SN-HPCs. Our results highlight, for the first time, CTNNB1 mutations as the likely initiating molecular events driving SN-HPC tumorigenesis, which places SN-HPC among the growing family of ß-catenin-driven mesenchymal neoplasms.

Phenotypical and molecular distinctness of sinonasal haemangiopericytoma compared to solitary fibrous tumour of the sinonasal tract.

AIMS: Sinonasal haemangiopericytoma (SN-HPC) is a rare sinonasal mesenchymal neoplasm of perivascular myoid cell origin. Solitary fibrous tumour (SFT) occurs only very rarely in the sinonasal tract. SFT and soft tissue HPC have been considered a single entity. Recently, recurrent gene fusions involving NAB2-STAT6 resulting in differential expression of STAT6 were characterized as central molecular events in SFT. However, no data exist for NAB2-STAT6 status or STAT6 expression in SN-HPC. METHODS AND RESULTS: We examined six SN-HPCs and two sinonasal SFTs by immunohistochemistry and RT-PCR for NAB2-STAT6 fusions. SN-HPC affected three females and three males (mean age: 72 years). They expressed smooth muscle actin, lacked strong CD34 reactivity and were negative for nuclear STAT6 expression. RT-PCR analysis confirmed the absence of NAB2-STAT6 fusions in all cases. Conversely, both sinonasal SFTs (in males aged 39 and 52 years) displayed classical features of pleuropulmonary and soft-tissue SFTs (uniformly CD34-positive with strong nuclear expression of STAT6). RT-PCR revealed NAB2-STAT6 fusions in both cases. CONCLUSIONS: These findings confirm the molecular and phenotypical distinctness of these two entities. While SN-HPC is a site-specific sinonasal neoplasm of as yet unknown molecular pathogenesis, sinonasal SFTs show phenotypical and molecular identity to their pleural/extrapleural counterparts.

Sensitive and specific detection of EML4-ALK rearrangements in non-small cell lung cancer (NSCLC) specimens by multiplex amplicon RNA massive parallel sequencing.

OBJECTIVES: Recurrent gene fusions of anaplastic lymphoma receptor tyrosine kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) have been recently identified in ∼5% of non-small cell lung cancers (NSCLCs) and are targets for selective tyrosine kinase inhibitors. While fluorescent in situ hybridization (FISH) is the current gold standard for detection of EML4-ALK rearrangements, several limitations exist including high costs, time-consuming evaluation and somewhat equivocal interpretation of results. In contrast, targeted massive parallel sequencing has been introduced as a powerful method for simultaneous and sensitive detection of multiple somatic mutations even in limited biopsies, and is currently evolving as the method of choice for molecular diagnostic work-up of NSCLCs. MATERIALS AND METHODS: We developed a novel approach for indirect detection of EML4-ALK rearrangements based on 454 massive parallel sequencing after reverse transcription and subsequent multiplex amplification (multiplex ALK RNA-seq) which takes advantage of unbalanced expression of the 5' and 3' ALK mRNA regions. Two lung cancer cell lines and a selected series of 32 NSCLC samples including 11 cases with EML4-ALK rearrangement were analyzed with this novel approach in comparison to ALK FISH, ALK qRT-PCR and EML4-ALK RT-PCR. RESULTS: The H2228 cell line with known EML4-ALK rearrangement showed 171 and 729 reads for 5' and 3' ALK regions, respectively, demonstrating a clearly unbalanced expression pattern. In contrast, the H1299 cell line with ALK wildtype status displayed no reads for both ALK regions. Considering a threshold of 100 reads for 3' ALK region as indirect indicator of EML4-ALK rearrangement, there was 100% concordance between the novel multiplex ALK RNA-seq approach and ALK FISH among all 32 NSCLC samples. CONCLUSION: Multiplex ALK RNA-seq is a sensitive and specific method for indirect detection of EML4-ALK rearrangements, and can be easily implemented in panel based molecular diagnostic work-up of NSCLCs by massive parallel sequencing.

Solitary fibrous tumors/hemangiopericytomas with different variants of the NAB2-STAT6 gene fusion are characterized by specific histomorphology and distinct clinicopathological features.

Recurrent somatic fusions of the two genes, NGFI-A-binding protein 2 (NAB2) and STAT6, located at chromosomal region 12q13, have been recently identified to be presumable tumor-initiating events in solitary fibrous tumors (SFT). Herein, we evaluated a cohort of 52 SFTs/hemangiopericytomas (HPCs) by whole-exome sequencing (one case) and multiplex RT-PCR (all 52 cases), and identified 12 different NAB2-STAT6 fusion variants in 48 cases (92%). All 52 cases showed strong and diffuse nuclear positivity for STAT6 by IHC. We categorized the fusion variants according to their potential functional effects within the predicted fusion protein and found strong correlations with relevant clinicopathological features. Tumors with the most common fusion variant, NAB2ex4-STAT6ex2/3, corresponded to classic pleuropulmonary SFTs with diffuse fibrosis and mostly benign behavior and occurred in older patients (median age, 69 years). In contrast, tumors with the second most common fusion variant, NAB2ex6-STAT6ex16/17, were found in much younger patients (median age, 47 years) and represented typical HPCs from deep soft tissue with a more aggressive phenotype and clinical behavior. In summary, these molecular genetic findings support the concept that classic pleuropulmonary SFT and deep-seated HPC are separate entities that share common features but correlate to different clinical outcome.

Effect of lung flooding and high-intensity focused ultrasound on lung tumours: an experimental study in an ex vivo human cancer model and simulated in vivo tumours in pigs.

BACKGROUND: High-intensity focused ultrasound is a valuable tool for minimally invasive tumour ablation. However, due to the air content in ventilated lungs, lung tumours have never been treated with high-intensity focused ultrasound. Lung flooding enables efficient lung sonography and tumour imaging in ex vivo human and in vivo porcine lung cancer models. The current study evaluates the effectiveness of lung flooding and sonography-guided high-intensity focused ultrasound for lung tumour ablation in ex vivo human and in vivo animal models. METHODS: Lung flooding was performed in four human lung lobes which were resected from non-small cell lung cancers. B-mode imaging and temperature measurements were simultaneously obtained during high-intensity focused ultrasonography of centrally located lung cancers. The tumour was removed immediately following insonation and processed for nicotinamide adenine dinucleotide phosphate-diaphorase and H&E staining. In addition, the left lungs of three pigs were flooded. Purified BSA in glutaraldehyde was injected centrally into the left lower lung lobe to simulate a lung tumour. The ultrasound was focused transthoracically through the flooded lung into the simulated tumour with the guidance of sonography. The temperature of the tumour was simultaneously measured. The vital signs of the animal were monitored during the procedure. RESULTS: A well-demarcated lesion of coagulation necrosis was produced in four of four human lung tumours. There did not appear to be any damage to the surrounding lung parenchyma. After high-intensity focused ultrasound insonation, the mean temperature increase was 7.5-fold higher in the ex vivo human tumour than in the flooded lung tissue (52.1 K ± 8.77 K versus 7.1 K ± 2.5 K). The transthoracic high-intensity focused ultrasound of simulated tumours in the in vivo model resulted in a mean peak temperature increase up to 53.7°C (±4.5). All of the animals survived the procedure without haemodynamic complications. CONCLUSIONS: High-intensity focused ultrasound with lung flooding produced a thermal effect in an ex vivo human lung carcinoma and in vivo simulated lung tumours in a porcine model. High-intensity focused ultrasound is a potential new strategy for treating lung cancer.

Increased detection rates of EGFR and KRAS mutations in NSCLC specimens with low tumour cell content by 454 deep sequencing.

Detection of activating EGFR mutations in NSCLC is the prerequisite for individualised therapy with receptor tyrosine kinase inhibitors (TKI). In contrast, mutant downstream effector KRAS is associated with TKI resistance. Accordingly, EGFR mutation status is routinely examined in NSCLC specimens, but the employed methods may have a dramatic impact on the interpretation of results and, consequently, therapeutic decisions. Specimens with low tumour cell content are at particular risk for false-negative EGFR mutation reporting by sequencing with Sanger chemistry. To improve reliability of detecting clinically relevant mutant variants of EGFR and KRAS, we took full advantage of 454 deep sequencing and developed a two-step amplification protocol for the analysis of EGFR exons 18-21 and KRAS exons 2 and 3. We systematically addressed the sensitivity, reproducibility and specificity of the developed assay. Mutations could be reliably identified down to an allele frequency of 0.2-1.5 %, as opposed to 10-20 % detection limit of Sanger sequencing. High reproducibility (0-2.1 % variant frequency) and very low background level (0.4-0.8 % frequency) further complement the reliability of this assay. Notably, re-evaluation of 16 NSCLC samples with low tumour cell content ≤40 % and EGFR wild type status according to Sanger sequencing revealed clinically relevant EGFR mutations at allele frequencies of 0.9-10 % in seven cases. In summary, this novel two-step amplification protocol with 454 deep sequencing is superior to Sanger sequencing with significantly increased sensitivity, enabling reliable analysis of EGFR and KRAS in NSCLC samples independent of the tumour cell content.

Sealing of pulmonary arteries with LigaSure: in vivo and ex vivo examinations.

OBJECTIVE: The LigaSure device has been demonstrated to be safe for systemic vessels up to 7 mm in diameter, although its use in thoracic surgery remains understudied. We aimed to evaluate the safety of LigaSure for pulmonary artery sealing. METHODS: In 30 cases of open lung lobectomy, 15 small pulmonary arteries (diameter, 3-5 mm) and 15 thick pulmonary arteries (diameter, 6-8 mm) were divided with LigaSure. Before closure of the thoracotomy, the vessel stumps were ligated proximal to the sealing zone, resected, and preserved in formaldehyde for histopathologic examination. In a control group, a similar number and size of pulmonary arteries were suture-ligated. The burst pressure of the pulmonary arteries from the resected lung lobes was measured. RESULTS: The mean burst pressure of small pulmonary arteries was 4.3-fold less after sealing than after ligation (315 ± 213.1 mm Hg vs 1345 ± 256 mm Hg; P < .001), and 6.4-fold less than after ligation of thick pulmonary arteries (156 ± 42.5 mm Hg vs 1007 ± 141.6 mm Hg; P < .001). Sealed pulmonary arteries >5 mm in diameter have a burst pressure that is 50% less than that of smaller arteries (P < .001). In all cases after sealing, the histologic examination demonstrated only a fusion of the adventitia, whereas the intima and media were replaced and invaginated into the vessel lumen. CONCLUSIONS: LigaSure does not result in complete fusion of the wall layers of pulmonary arteries. The pulmonary artery burst pressure after sealing is significantly less compared with conventional suture ligation. It remains unclear whether these findings create a clinical risk of rupture.

Cystic medionecrosis of the carotid bifurcation as an uncommon cause of stroke.

BACKGROUND AND PURPOSE: Cystic medionecrosis is characterized by degeneration of elastic and collagenous fibers in the media and predominantly involves the thoracic aorta. This rare disease is usually manifested clinically by a dissecting aneurysm. Cystic medionecrosis as a cause of moderate stenosis of the carotid artery in a patient having a stroke has not been reported. SUMMARY OF CASE: We report a case of a man who had a cerebral infarction caused by medium-degree stenosis of the left internal carotid artery. Duplex sonography and magnetic resonance imaging revealed no typical signs of dissection. The stenosis was caused by cystic medionecrosis that involved only the carotid bifurcation with microdissection, predominantly older intramural hemorrhage, and fresh intraluminal thrombotic deposits. CONCLUSIONS: Patients with cystic medionecrosis may have a stroke due to short-track stenosis of the internal carotid artery.

Effects of radiofrequency ablation on normal lung tissue in a swine model.

RATIONALE AND OBJECTIVES: To determine the effect of radiofrequency (RF) ablation on normal lung tissue in an animal model. MATERIALS AND METHODS: RF ablation of lung tissue was performed on eight swine under computed tomographic control. Group A (n = 4) received peripheral ablation (subpleural needle placement) and group B (n = 4) received central ablation (hilar needle placement). RF ablation was applied via a single 4.5-gauge internally cooled electrode with a 2-cm tip for 12 minutes. The ablation was monitored with computed tomography at 3, 7, and 12 minutes, and 10 minutes after ablation. After 3, 7, 40, and 60 days, computed tomography was performed, and the animals were sacrificed to examine the lung tissue both macroscopically and histopathologically. RESULTS: There were no deaths from RF ablation. In group A, coagulative necrosis was resorbed almost completely and transformed into a fibrotic scar after 60 days. No pneumothorax, pleural effusion, or lung abscess was observed. In group B, there was also a transformation of the necrosis into connective tissue. Neither the pulmonary vessels nor the bronchi of the hilum abutting the coagulative necrosis were damaged. After 60 days, no vascular thrombosis, bleeding, aneurysm, bronchial stenosis, or bronchopulmonary fistula was observed. CONCLUSION: RF ablation of lung tissue affects coagulation necrosis, causing scar transformation. There was no damage to either great vessels or bronchi. The application of RF ablation for tumors located in or near functional structures appears feasible without severe complications.

Dynamic MRI and CAD vs. choline MRS: where is the detection level for a lesion characterisation in prostate cancer?

PURPOSE: To evaluate the role of pre-interventional fused high resolution T2-weighted images with parametrically analysed dynamic contrast enhanced T1-weighted magnetic resonance (MR) images (DCE-MRI) and 1H magnetic resonance spectroscopy (MRS) for a precise biopsy for the detection of prostate cancer and for the delineation of intraprostatic subvolumes for intensity modulated radiation therapy (IMRT). INCLUSION CRITERIA: Pathological prostate-specific antigen values (PSA) and/or previously negative transrectal ultrasound guided biopsy. Standardised biopsy of the prostate divided into 20 regions. Image fusion of coloured parametric maps derived from DCE-MRI and MRS (single voxel spectroscopy, SVS; chemical shift imaging, CSI) with T2 images for morphological localisation using the MR-workstation, a separate CAD-workstation (CAD: computer aided diagnosis) or a radiation treatment planning system. Correlation of these intraprostatic subvolumes with histology and cytokeratin-positive areas in prostatectomy species. RESULTS: DCE-MRI: Sensitivity 82%, specificity 89%, accuracy 88%, positive predictive value 61%, negative predictive value 96%. SVS: Sensitivity 55%, specificity 62%. CSI: Sensitivity 68%, specificity 67%. False positive findings due to prostatitis, adenomatous hyperplasia, false negative findings due to low signal (PIN (prostatic intraepithelial neoplasia), cut-off level for DCE-MRI: lesions smaller 3 mm and less than 30% cancer cells, for SVS: lesions smaller 8 mm and less than 50% cancer cells), for CSI: lesions smaller 4 mm and less than 40% cancer cells. Our MR data are correlated with published choline PET/CT data (PET/CT: hybrid scanner of positron emission tomography and computed tomography). CONCLUSIONS: DCE-MRI and MRS are helpful for a precise biopsy of the prostate. The European Society for Therapeutic Radiology and Oncology (ESTRO) guidelines 2006 for radiation treatment planning of the prostate have to be revised, if the standardised biopsy will be replaced by a lesion-orientated biopsy. Until now it is unclear, if the parametric maps of DCE-MRI and MRS can be used for radiation treatment planning of the prostate.

Sporadic and radiation-associated papillary thyroid cancers can be distinguished using routine immunohistochemistry.

We investigated protein abundance in order to differentiate radiation-associated papillary thyroid cancers (PTC) from other etiologies for e.g. forensic purposes. Proteins were extracted from frozen tissues originating from 91 sporadic PTCs and 86 post-Chernobyl PTCs. Proteins were separated gel-electrophoretically, gels were silver stained, spots scanned and their intensity quantified. After excision of spots from the gel and protein digestion, MALDI-TOF mass spectrometry was performed followed by correlation of these results to human proteins using appropriate software and database. After this screening approach, altogether 20 candidate proteins were selected and measured semiquantitatively (Remmele score) using immunohistochemistry. Logistic regression modeling was performed for discriminating the groups. NTRK1, metalloproteinases (MMP-1, MMP-9 and MMP-13) and Cathepsins (-W and -X) proved to be of highest significance for discriminating the groups irrespective of the regression model utilized. When considering age and gender, each of 3 proteins by itself made possible a complete separation of the groups otherwise a combination of 2 of the 5 proteins mentioned was needed. In conclusion, abundance of proteins known to be associated with a more aggressive tumor type (MMPs and Cathepsins) appeared increased in post-Chernobyl PTC compared to sporadic PTC, thus underlining the known aggressiveness of radiation-associated PTC. These proteins make it possible to completely distinguish post-Chernobyl from sporadic PTC using routine immunohistology.

Expression of c-MET, low-molecular-weight cytokeratin, matrix metalloproteinases-1 and -2 in spinal chordoma.

AIMS: In skull base chordoma, c-MET expression has been reported to correlate with younger patient age and favourable prognosis; however, it also contributes to tumour invasiveness, especially in recurrent lesions, suggesting variable roles for c-MET according to clinical status. The aim of this study was to investigate the significance of c-MET expression in spinal chordoma, which affects patients who are 10-20 years older than those with skull base chordoma. METHODS AND RESULTS: Using immunohistochemical techniques, the expression of c-MET and its ligand, hepatocyte growth factor (HGF) was investigated in 34 primary spinal chordomas and compared with other clinicopathological parameters. Expression of c-MET and HGF was observed in 85.3 and 21.7% of lesions, respectively. c-MET expression correlated with the expression of an epithelial marker, low-molecular-weight cytokeratin (CAM5.2). Lesions with higher c-MET expression showed significantly stronger expression of proteinases, including matrix metalloproteinase (MMP)-1 and MMP-2. However, c-MET expression was not associated with patient age, proliferative ability estimated by MIB-1 labelling index, or prognosis. CONCLUSIONS: c-MET expression was observed in most spinal chordomas and correlated with the expression of CAM5.2, suggesting a relationship to an epithelial phenotype.

Experimental studies on a new highly porous hydroxyapatite matrix for obliterating open mastoid cavities.

OBJECTIVE: In an initial preliminary study, the applicability of a new high-porosity hydroxyapatite (HA) ceramic for obliterating large open mastoid cavities was proven and tested in an animal model (bulla of guinea pig). STUDY DESIGN: Experimental study. METHODS: NanoBone, a highly porous matrix consisting of 76% hydroxyl apatite and 24% silicone dioxide fabricated in a sol-gel technique, was administered unilaterally into the opened bullae of 30 guinea pigs. In each animal, the opposite bulla was filled with Bio-Oss, a bone substitute consisting of a portion of mineral bovine bone. Histologic evaluations were performed 1, 2, 3, 4, 5, and 12 weeks after the implantation. RESULTS: After an initial phase in which the ceramic granules were surrounded by inflammatory cells (1-2 wk), there were increasing signs of vascularization. Osteoneogenesis and-at the same time-resorption of the HA ceramic were observed after the third week. No major difference in comparison to the bovine bone material could be found. DISCUSSION: Our results confirm the favorable qualities of the new ceramic reported in association with current maxillofacial literature. Conventional HA granules used for mastoid obliteration to date often showed problems with prolonged inflammatory reactions and, finally, extrusions. In contrast to those ceramics, the new material seems to induce more osteoneogenesis and undergoes early resorption probably due to its high porosity. Overall, it is similar to the bovine bone substance tested on the opposite ear in each animal. Further clinical studies may reveal whether NanoBone can be an adequate material for obliterating open mastoid cavities in patients.

Expression of hepatocyte growth factor and c-MET in skull base chordoma.

BACKGROUND: Hepatocyte growth factor (HGF) is a multipotent cytokine that is mediated by its receptor, c-MET. HGF/c-MET contributes to tumor progression in many human malignancies; however, HGF/c-MET is inversely correlated with aggressive biologic behavior in other cancers. Conversely, to the authors' knowledge, little is known regarding the significance of HGF/c-MET expression in skull base chordoma. METHODS: Using immunohistochemical techniques, the authors investigated HGF/c-MET expression in 46 primary and 25 recurrent lesions, and compared it with the expression of proteinases and cell differentiation markers, proliferative ability, and other clinicopathologic parameters. RESULTS: c-MET was found to be expressed in 70.0% of primary and 88.0% of recurrent lesions. HGF expression was scarcely detected. Higher c-MET expression was found to be correlated with younger patient age. Lesions with a higher expression of low molecular weight cytokeratin (CAM5.2) demonstrated significantly higher c-MET scores in both primary and recurrent lesions compared with those with lower CAM5.2 expression. In recurrent lesions, higher c-MET expression was found to be associated with the scores of matrix metalloproteinase (MMP)-1, MMP-2, tissue inhibitor of matrix metalloproteinase-1, and urokinase plasminogen activator (uPA); however, only uPA was found to be correlated with higher c-MET expression in primary lesions. c-MET expression did not appear to be correlated with MIB-1 labeling index. Patients with higher c-MET expression were found to have longer survival. CONCLUSIONS: In the current study, c-MET expression was a common event, and was found to be correlated with CAM5.2 expression, younger patient age, and a favorable prognosis in patients with skull base chordoma. However, HGF/c-MET paracrine signaling also may contribute to its invasive ability, especially in recurrent lesions.

Expression of matrix metalloproteinases-1, -2, and -9; tissue inhibitors of matrix metalloproteinases-1 and -2; cathepsin B; urokinase plasminogen activator; and plasminogen activator inhibitor, type I in skull base chordoma.

Little is known about proteinase expression in skull base chordoma, a rare bone tumor exhibiting local invasiveness. Using immunohistochemical techniques, we investigated the expression of matrix metalloproteinases (MMPs)-1, -2, and -9; tissue inhibitors of matrix metalloproteinases (TIMPs)-1 and -2; cathepsin B (CatB); urokinase plasminogen activator (uPA); and plasminogen activator inhibitor, type I (PAI1), in 45 patients with skull base chordoma (45 primary and 25 autologous recurrent lesions). We compared these data with clinicopathologic parameters and the expression of cell differentiation markers. MMP-1, MMP-2, TIMP-1, CatB, uPA, and PAI1 were frequently expressed, and there was a significant correlation in the expression of some proteinases. Immunoreactivity for MMP-1, MMP-2, CatB, and uPA was significantly higher in lesions exhibiting tumor infiltration of host bone than in those without such components. Expression of MMP-1, TIMP-1, CatB, and uPA was associated with that of low-molecular-weight cytokeratin (CAM5.2). There were no differences in proteinase expression in 25 pairs of primary and their recurrent lesions, and proteinase expression did not predict local recurrences. However, patients with higher expression of both MMP-1 and uPA showed worse prognosis compared with the others. In conclusion, expression of some proteinases correlated with CAM5.2 expression and seemed to play an important role in a synergistic manner in the invasion process in skull base chordoma. The authors believe that elevated expression of MMP-1 and uPA can be used to identify patients with a worse prognosis in skull base chordoma.

Laser scanning microscopy of the human larynx mucosa: a preliminary, ex vivo study.

OBJECTIVE: Laser scanning microscopy (LSM) supplies in vivo information from epithelia up to depths between 0.1 to 0.5 mm. The aim of this ex vivo prospective pilot study was to investigate the potential use of LSM for the diagnosis of laryngeal cancer and its precursors. METHODS: Forty-three larynx specimens of 26 patients (age 35-61 years, mean age 51.9+/-9.5 years; 7 women and 19 men) with laryngeal lesions were investigated with LSM. The LSM findings were compared with histopathologic sections. The following criteria were used for characterization of cancerous lesions: enlarged nuclei, enlarged cells with variable shapes, cluster of cells, increased nucleus/cytoplasm ratio, irregular cell architecture, and loss of cellular junctions characterized by lack of visualization of the cell membrane. RESULTS: LSM enables the visualization of epithelium up to the basement membrane, Reincke space, the subepithelial vessels, and the fibers of the subepithelial space. In contrast to the squamous epithelium, the respiratory epithelium bears kinocilia. The beat of the cilia and the directed mucous transport can be observed ex vivo. With the use of the presented malignancy criteria, a sensitivity of 72.7% and a specificity of 82.9% for differentiation of dysplasia and benign laryngeal lesions from cancer were reached. CONCLUSIONS: LSM in an ex vivo manner supplies microscopic images up to the subepithelial space. LSM could represent a new technology in laryngology to visualize larynx epithelia. In the next step, in vivo LSM will be applied to evaluate laryngeal lesion in vivo.

Diversity of CD97 in smooth muscle cells.

CD97, an epidermal growth factor (EGF)-TM7 receptor, is not restricted to hematopoetic and carcinoma cells but is also found on smooth muscle cells (SMC). We have examined its location and biochemical structure in various normal and tumorigenic SMC-containing tissues. SMC of the urinary bladder, lung bronchi and bronchioles, myometrium, and gastrointestinal tract were immunohistologically stained by using monoclonal antibodies (mabs) to the CD97 stalk region (CD97(stalk)). Mabs directed against an N-glycosylation-dependent epitope within the EGF-domains (CD97(EGF)) did not bind to normal SMC. Vascular SMC, which was also CD97(EGF)-negative, showed further CD97 heterogeneity. Only a few, if any, SMC from the aorta or elastic arteries of the systemic circulation were positive for CD97 mRNA and therefore also for CD97(stalk). CD97(stalk)-positive SMC were slightly more numerous in muscular and peripheral arteries. In contrast, most venous SMC expressed CD97(stalk). A comparison with other SMC molecules revealed a similar but not identical staining pattern for CD97(stalk) and desmin. Further CD97 heterogeneity was observed during SMC transformation. All leiomyomas (n=5) and nine out of 21 leiomyosarcomas were positive for both CD97(stalk) and CD97(EGF). As expected, CD97(EGF)-positive SMC tumors expressed partly N-glycosylated CD97. Seven out of 21 leiomyosarcomas were completely devoid of CD97. Thus, CD97 showed variable expression in vascular and biochemical modification in tumorigenic SMC, suggesting that the function of the molecule is specific for the SMC subtype.