[Show abstract][Hide abstract] ABSTRACT: Granulomatous inflammations, characterized by the presence of activated macrophages (MAs) forming epithelioid cell (EPC) clusters, are usually easy to recognize. However, in ambiguous cases the use of a MA marker that expresses selectively in EPCs may be needed. Here, we report that carboxypeptidase-M (CPM), a MA-differentiation marker, is preferentially induced in EPCs of all granuloma types studied, but not in resting MAs. As CPM is not expressed constitutively in MAs, this allows utilization of CPM-immunohistochemistry in diagnostics of minute granuloma detection when dense non-granulomatous MAs are also present. Despite this rule, hardly any detectable CPM was found in advanced/active tubercle caseous disease, albeit in early tuberculosis granuloma, MAs still expressed CPM. Indeed, in vitro both the CPM-protein and -mRNA became downregulated when MAs were infected with live mycobacteria. In vitro, MA-CPM transcript is neither induced remarkably by interferon-γ, known to cause classical MA activation, nor by IL-4, an alternative MA activator. Instead, CPM is selectively expressed in lipid-laden MAs, including the foam cells of atherosclerotic plaques, xanthomatous lesions and lipid pneumonias. By using serum, rich in lipids, and low-density lipoprotein (LDL) or VLDL, CPM upregulation could be reproduced in vitro in monocyte-derived MAs both at transcriptional and protein levels, and the increase is repressed under lipid-depleted conditions. The microarray analyses support the notion that CPM induction correlates with a robust progressive increase in CPM gene expression during monocyte to MA maturation and dendritic cell (DC) differentiation mediated by granulocyte-MA-colony-stimulating factor+IL-4. M-CSF alone also induced CPM. These results collectively indicate that CPM upregulation in MAs is preferentially associated with increased lipid uptake, and exposure to CSF, features of EPCs, also. Therefore, CPM-immunohistochemistry is useful for granuloma and foam MA detections in tissue sections. Furthermore, the present data offer CPM for the first time to be a novel marker and cellular player in lipid uptake and/or metabolism of MAs by promoting foam cell formation.
[Show abstract][Hide abstract] ABSTRACT: The current study was done as a validation study prior to setting up a clinical HER2 testing service using the new commercial Poseidon HER2 fluorescence in situ hybridisation (FISH) assay. However, it was felt that the experience of the authors of this study may be of interest to other laboratories when considering setting up a HER2 diagnostic facility.
122 patients who had been diagnosed with invasive breast cancer were selected. Immunolabelling with HercepTest, PathVysion and Poseidon FISH assays were carried out using tissue microarray blocks.
Concordance correlation coefficients showed near perfect agreement in average HER2 and centromere specific signal counts per cell and in the HER2/CEP17 ratios between the PathVysion and the Poseidon FISH assays. In addition, the kappa measure showed perfect agreement (kappa 0.9441, p<0.0001), and if only 2+ cases were considered there was substantial agreement (kappa 0.7671, p=0.0006), between the two assays. The sensitivity and the specificity of the Poseidon FISH kit were calculated to be 95.2% and 100%, respectively, whereas the positive predictive value (PPV) and negative predictive value (NPV) were 100% and 99%, respectively. With regard to the ability to presume HER2 polysomy, the Poseidon FISH kit had a sensitivity of 93.3% and a specificity of 99.1%, with PPV and NPV of 93.3% and 99.1%, respectively, as assessed with PathVysion classification as the reference.
Statistical analysis confirmed that the two FISH assays are comparable in terms of detection of HER2 gene amplification. Proceeding from these findings, the genetic diagnoses obtained with the Poseidon kit can be considered to be as valuable as the results from the Food and Drug Administration approved PathVysion assay, and its utilisation in routine HER2 diagnostics is proposed.
Journal of clinical pathology 12/2009; 63(4):341-6. · 2.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The alpha-synuclein immunopositive and chaotrope-insoluble material from human brains with Lewy body pathology was analyzed by mass spectrometry. From the proteinase K-cleavable peripheral fraction of Lewy bodies, which was densely cross-linked by gamma-glutamyl-epsilon-lysine bonds between HspB1 and ubiquitin in a pattern similar to neurofibrillary tangles (Nemes, Z., Devreese, B., Steinert, P. M., Van Beeumen, J., and Fésüs, L. (2004) FASEB J. 18, 1135-1137), 53 proteins were identified. In the core of Lewy bodies only alpha-synuclein was found, and it contained a low amount of intramolecular cross-links between Gln-99 and Lys-58. In vitro cross-linking of alpha-synuclein by transglutaminases 1-3 and 5 produced a heterogeneous population of variably cross-linked alpha-synucleins in solution, which inhibited the aggregation of the protein into amyloid. However, in the presence of phosphatidylserine-rich membranes and micromolar calcium concentrations, the cross-linking by transglutaminases 1, 2, and 5 showed specificity toward the utilization of Gln-99 and Lys-58. As shown by thioflavin T fluorescence monitoring, the formation of this cross-link accelerated the aggregation of native alpha-synuclein. Chemical cross-linking of residues 58-99 triggered amyloid formation, whereas such bonding of residues 99 to 10 was inhibitory. Our findings reveal the pivotal role of membrane attachment and transglutaminase-mediated intermolecular cross-linking for the propagative misfolding and aggregation of alpha-synuclein.
Journal of Biological Chemistry 09/2009; 284(40):27252-64. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Since the first publication of a rhabdoid cancer, described as an infrequent variant of Wilms' tumor, several cases of extrarenal rhabdoid tumor have been reported in the literature. Here, we report on a primary rhabdoid cancer of the small intestine, and give a review of the data available in the literature. An 81-year-old male patient was admitted to the Department of Internal Medicine with subileus and bloody stools. While gastroscopy and colonoscopy failed to identify a neoplastic disease, abdominal US and CT raised the suspicion of a malignancy involving the descending colon. Ileus-mandated laparotomy disclosed an obstructive tumor of the ileum. This segment was resected, followed by several relapses, and the patient died after a 7-month-period of non-relenting deterioration. Histological work-up of the numerous biopsies disclosed a cellular, solid, necrotic, hemorrhagic, and invasive tumor. The cancer cells were round-to-polygonal, and scattered bizarre pleomorphic cells with prominent nucleoli were common. The overall appearance of the neoplasm was highly similar in every specimen. The immunohistochemical phenotype of the malignant cells indicated rhabdoid characteristics. A thorough search of the literature revealed additional 22 cases of primary rhabdoid GI cancers. This report aims to call the pathologist's attention to the differential diagnostic importance of this entity.
Pathology - Research and Practice 05/2009; 206(2):110-5. · 1.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Enhanced expression of transglutaminases is a frequent, though not obligatory phenomenon in apoptosis, which is associated with cells dying in steady interaction with their tissue environment. Modification of cellular proteins by transglutamination is a tightly controlled procedure. If transglutamination is dysregulated, it has profound and potentially detrimental consequences on cellular functioning. Under conditions normally occurring in living cells transglutaminase activity is usually undetectably low (latent) and can only be tested by careful preselection of proteins of interest. In late stages of apoptosis, transglutaminases can become rampant in dying cells and a minuscule fraction of dead cells may overshadow many more living ones, which may cause inherent and severe methodological and interpretation bias. Therefore, in this chapter, we describe the analysis of dead cell remnants for protein-bound transglutaminase-mediated cross-link content. In the techniques described below, we rely on the increasing availability and user-friendliness of mass spectrometric equipments.
[Show abstract][Hide abstract] ABSTRACT: The overexpression of HER-2/neu is an independent prognostic factor of clinical outcome of breast cancer, therefore determination of HER-2/neu status is now an integral part of the clinicopathologic workup. The ways of measuring the copy number of the HER-2/neu gene in tumor cells comprise in situ hybridization techniques and real-time polymerase chain reaction (PCR). Quantitative real-time PCR is a relatively new technique for assessing HER-2/neu gene amplification with high sensitivity. However, the HER-2/neu Quantification Kit developed by Roche designed for a LightCycler 1.5 platform had been withdrawn from the commercial market; therefore, we were encouraged to design an alternative LightCycler-based method that offers the desired level of reliability. One hundred breast cancer cases with known HER-2/neu status have been examined with the original Roche developed HER-2/neu Quantification kit and the custom real-time PCR assay. The newly developed, custom PCR showed sensitivity of 91.43%, specificity of 90.63%, and accuracy of 90.91% taking fluorescence in situ hybridization results as the end point. We have described a novel real-time PCR technique for the relative quantification of the HER2/neu gene on a LightCycler 1.5 platform. We have determined that our method is eligible and ideal for the supplement of regular fluorescence in situ hybridization reactions, concerning its high sensitivity and reliability.
Applied immunohistochemistry & molecular morphology: AIMM / official publication of the Society for Applied Immunohistochemistry 01/2009; 17(3):247-54. · 1.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gefitinib and erlotinib are both selective EGFR tyrosine kinase inhibitors (EGFR-TKIs) that have produced responses in a small subgroup of lung cancer patients. The strongest evidence for a role of EGFR in the biology of glioblastoma stems from clinical trials in which 15-20% of recurrent glioblastoma patients experienced significant tumour regression in response to these small-molecule EGFR kinase inhibitors. We examined the protein-kinase domain of the EGFR gene, EGFR protein expression and EGFR gene amplification in 20 cases of recurrent GBMs. EGFR protein over-expression was found in 65% of cases. EGFR protein over-expression was associated with EGFR gene amplification in 35% of cases, and with high polysomy in 15% of cases. No mutations were found in the TK domain of the EGFR gene. Our results confirm that mutations in the kinase domain are absent in recurrent GBM, and this might be a preponderant factor in the lack of major clinical responses of TKIs in GBM, recent studies have suggested that responsiveness to EGFR kinase inhibitors was strongly associated with coexpression of EGFRvIII and PTEN. Further prospective validation of EGFRvIII and PTEN as predictors of the clinical response to EGFR kinase inhibitors in recurrent GBM is strongly anticipated.
Pathology & Oncology Research 09/2008; 15(2):225-9. · 1.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Carboxypeptidase-M (CPM) is a membrane-bound peptidase that metabolizes peptides, and is present in pneumocytes. CPM hydrolyses the C-terminal arginine of epidermal growth factor (EGF) resulting in des-Arg53-EGF which binds to the EGF receptor (EGFR) with an equal or greater affinity than native EGF. Therefore, this study focused on the possible presence of CPM in human lung adenocarcinomas (ADC) and evaluated the relationship between CPM and EGFR by assessing the impact of expressions on patient clinical outcome.
This is a retrospective study of 110 patients who underwent resection of the primary tumour (92) or metastatic tissues (18) for treatment or diagnosis. Immunohistochemistry (IHC) for CPM and EGFR was made in serial sections using standard methods.
This study demonstrates for the first time that 23.6% of ADCs express carboxypeptidase-M (26/110), mainly in membrane-bound forms. The amounts and the extent of CPM within tumours vary from low levels to obviously overexpressed forms. The immunohistochemical positivity (+) for CPM in ADCs negatively correlated with disease survival. In addition, 80% of CPM+ adenocarcinomas (21/26) showed a coexpression with EGFR suggesting a high prevalence for coexistence. The follow up data indicated a significantly shorter 5-year survival time for patients with CPM+-EGFR+ (double-positive) tumours compared to those harbouring neoplasias negative for both proteins (9.5 vs. 60.4% survivals, P < 0.001).
The fact that CPM+ ADCs often co-express with EGFR suggests a functional-regulatory link between these proteins which might have therapeutical consequences. The present novel data could lead to improved IHC tests in lung adenocarcinomas for EGFR expression.
Journal of Cancer Research and Clinical Oncology 04/2008; 134(4):439-51. · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Overexpression and/or gene amplification of the HER2 oncogene predicts worse prognosis and altered sensitivity to chemotherapy. Trastuzumab is capable of improving prognosis of HER2-positive breast cancer, but for the success of treatment appropriate HER2 testing is essential. Our aim was to determine the value of immunohistochemical (IHC) screening prior to fluorescence in situ hybridization (FISH). We assessed five conventional IHC assays (NCL-CB11, Pathway CB11, CBE356, CBE1, HercepTest) and the novel rabbit monoclonal antibody, RM-4B5, combined with FISH on 199 invasive breast cancer cases. Taking FISH as the endpoint, we calculated sensitivity, specificity, positive and negative predictive values (PPV, NPV) and accuracy for all IHC assays with either taking both 2+/3+ cases or only 3+ cases as IHC positives. With 2+/3+ cases HercepTest showed 100% sensitivity and NPV, while the highest specificity, PPV and accuracy was associated with RM-4B5 (97.36, 80 and 95.34%, respectively). The second highest values belonged to either NCL-CB11 or Pathway CB11. When calculating only with 3+ cases, the results were reversed with increased specificity, PPV and accuracy. Our findings suggest that improving sensitivity by using two parallel IHC reactions might be beneficial; we recommend primarily HercepTest and Pathway CB11. Nevertheless, we may consider performing FISH analysis without prior IHC screening.
[Show abstract][Hide abstract] ABSTRACT: MCF-7 cells undergo autophagic death upon tamoxifen treatment. Plated on non-adhesive substratum these cells died by anoikis while inducing autophagy as revealed by monodansylcadaverine staining, elevated light-chain-3 expression and electron microscopy. Both de novo and anoikis-derived autophagic dying cells were engulfed by human macrophages and MCF-7 cells. Inhibition of autophagy by 3-methyladenine abolished engulfment of cells dying through de novo autophagy, but not those dying through anoikis. Blocking exposure of phosphatidylserine (PS) on both dying cell types inhibited phagocytosis by MCF-7 but not by macrophages. Gene expression profiling showed that though both types of phagocytes expressed full repertoire of the PS recognition and signaling pathway, macrophages could evolve during engulfment of de novo autophagic cells the potential of calreticulin-mediated processes as well. Our data suggest that cells dying through autophagy and those committing anoikis with autophagy may engage in overlapping but distinct sets of clearance mechanisms in professional and non-professional phagocytes.
Cell Death and Differentiation 07/2007; 14(6):1117-28. · 8.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To study histopathologic changes and the role of the microglia/macrophage cell in the therapeutic effect of I-125 interstitial brachytherapy on the cerebral gliomas.
Out of a series of 60 cases with cerebral astrocytomas and other brain tumors treated with I-125 interstitial brachytherapy, autopsy materials were available in ten cases 0.75 and 60 months after irradiation. The patients were treated with the maximum dosage (60 Gy) on the tumor periphery. Besides the routine hematoxylin-eosine and Mallory's PTAH trichrome staining, immunohistochemical reactions were carried out for CD15, CD31, CD34, CD45, CD68, CPM, HAM56 and HLR-DR antigens on paraffin sections to study immunologic phenotypic characteristics of the reaction cell population around gliomas after I-125 treatment.
One month after irradiation, a necrotic zone developed around the I-125 seeds within the 72 Gy isodose curve. Histologically, there was a fresh coagulation necrosis in the center of the lesion. Reactive zone has not yet developed but scattered interstitial and perivascular CD68 positive macrophages were present in the surrounding brain tissues. Six months after the I-125 isotope treatment, a reactive zone developed: a microglial rim around the necrosis tissue, and a broad area of proliferating vessels and glial fibrillary acidic protein (GFAP) positive astroglial cells which contained CD68 positive activated microglial and macrophage cells. Fifty-four months after I-125 interstitial irradiation, the necrotic center became colliquative and cystic. The microglial rim was replaced by round end stage (HLR-DR and CD31 positive) macrophages. The reactive zone was characterized by astrocytic gliosis but vascular proliferation and macrophages were lacking.
Results of the present immunohistochemical study suggest that the early lesions are characterized by migrating macrophages apparently concerned with the removal of necrotic debris. The established phase of reactive zone around the necrotic center is characterized by a narrow inner rim of microglial accumulation and a broad outer area characterized by astrocytic gliosis, vascular proliferation, activated microglia and infiltration by macrophages. In the burned-out phases of I-125 interstitial brachytherapy of gliomas, the necrosis undergoes liquefaction and the microglial rim is replaced by astrocytic gliosis which can be considered as equivalent to the scar tissue formed around necrosis outside the central nervous system.
Neurological Research 05/2007; 29(3):233-8. · 1.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent cytogenetic studies have shown that reciprocal translocation t (17;22)(q22;q13) and a supernumerary ring chromosome derived from the translocation r(17;22) are highly characteristic of dermatofibrosarcoma protuberans (DFSP). The chromosomal rearrangements fuse the collagen type Ialpha1 (COL1A1) and the platelet-derived growth factor B-chain (PDGFB) genes. The COL1A1-PDGFB fusion transcript has been shown not only in conventional DFSP but also in a small series of DFSP with fibrosarcomatons areas (DFSP-FS) using reverse transcriptase-based conventional polymerase chain reaction. Nothing is known about the status of the COL1A1-PDGFB chimaeric gene in the pleomorphic areas of DFSP-PleoSarc (formerly known as DFSP-malignant fibrous sarcoma).
To show the COL1A1-PDGFB fusion transcript in transformed malignant fibrous histiocytoma.
A real-time polymerase chain reaction assay for the COL1A1-PDGFB fusion transcript in a series of DFSP containing sarcoma was conducted to determine whether the chimaeric gene could be identified in both components of DFSP-FS and DFSP-PleoSarc. Eight cases were analysed.
In seven cases, transcriptable RNA was detected, and in these cases, translocations were found between COL1A1 and PDGFB genes involving exons 27, 32, 34, 40 and 47 of the COL1A1 gene and exon 2 of the PDGFB gene.
From a diagnostic aspect, this assay can be particularly useful in confirming the diagnosis of sarcomatous DFSP. On the other hand, the COL1A1-PDGFB fusion gene was shown in three cases of DFSP containing pleomorphic sarcoma, which supports the theory of the common histogenesis.
Journal of Clinical Pathology 03/2007; 60(2):190-4. · 2.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tissue microarrays (TMAs) as current medical research tools significantly lower the costs of immunohistochemical examinations (IHC) and fluorescence in situ hybridization (FISH) while enabling high levels of standardization and reliability. Taking HER2 testing of breast cancer into consideration, we assessed the routine applicability of TMAs. A hundred and seventy-four consecutive samples of invasive breast cancer cases were selected. TMAs were constructed in order to conduct double HER2 immunohistochemical analysis and FISH abreast using the conventional slide by slide method. Comparing the immunohistochemical data obtained from TMAs with the routinely processed large sections, we found a 94.5%/92.7%, 85.7%/88.9% and 91.2%/90% concordance at immunohistochemically HER2-negative, HER2 2+ and 3+ cases using the CB11/HercepTest, respectively. FISH performed on TMAs helped to determine Herceptin therapy suitability in all cases, and when discordance was found, we controlled FISH on "large sections". Being able to conduct FISH examinations at a reasonable price with or without prior immunohistochemical analysis, departments confronted with a certain frequency of breast cancer cases might extensively use the type of TMAs applied in our study. This is a relieve not only with regard to diagnostic work using microarrays, but this also allows to take new directions in research by shedding light on certain unusual cases.
Pathology - Research and Practice 02/2007; 203(3):169-77. · 1.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to investigate histopathological changes and the role of the microglia/macrophage cell system in the therapeutic effect of iodine-125 (125I) interstitial brachytherapy on cerebral gliomas. Out of a series of 60 cases harboring cerebral astrocytomas and other brain tumors treated with 125I interstitial brachytherapy, autopsy material was available in 10 cases between 0.75 and 60 months after irradiation. The patients were treated with 60-Gy maximum doses at the tumor periphery. Besides the routine HE and Mallory's PTAH trichrome staining, immunohistochemical reactions were carried out for CD15, CD31, CD34, CD45, CD68 (PG-M1), CPM, HAM 56 and HLA-DR antigens to study immunological characteristics of the reactive cell population around gliomas after 125I treatment. The present immunohistochemical study demonstrated that the early lesions following 125I interstitial brachytherapy of gliomas are characterized by migrating macrophages apparently concerned with the removal of necrotic debris. The established phase of reactive zone around the necrotic center disclosed a narrow inner rim of microglial accumulation, and a broad outer area consisting of astrocytic gliosis, vascular proliferation, activated microglia and infiltration by macrophages. In the burned-out phase, the necrosis undergoes liquefaction, the microglial rim is replaced by end-stage macrophages, and the reactive zone is transformed into astrocytic gliosis, which can be considered as equivalent to scar tissue formed around necrosis outside of the central nervous system.
Progress in neurological surgery 02/2007; 20:312-23.
[Show abstract][Hide abstract] ABSTRACT: The composition of reactive cell populations, which constitute the majority of tumor load in Hodgkin's lymphoma (HL), can influence the prognosis of the disease. Besides widely accepted and applied prognostic scores, the authors evaluate biological factors that may have a prognostic impact. Previous data indicate that the rate of eosinophils and mast cells in the reactive cell population, determined already at diagnosis, can be used for this purpose. Histological samples from 104 patients with HL with an average follow-up period of 110 (24-214) months were retrospectively analyzed. Mast cell positivity was associated with better overall survival, although this difference was only of borderline statistical significance (p=0.092). No significant difference was found in parameters like overall survival (OS, p=0.906) or event-free survival (EFS, p=0.307) of eosinophil-positive vs. -negative cases or in EFS (p=0.742) of mast cell-positive vs. -negative individuals (criterion for a positive specimen was more than 5% of appropriate cells in the reactive cell population). Looking at the effect of eosinophilia and mastocytosis together, there was no significant difference between the subgroups categorized according to the combined presence of the two cell types. It seems that tissue eosinophil and mast cell predominance have no prognostic value that could be used in clinical practice, although a tendency for correlation of mast cell positivity with overall survival could be seen. For a definitive statement, multicenter studies should be performed involving a higher number of patients suffering from HL.
Pathology & Oncology Research 02/2007; 13(3):237-42. · 1.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The overexpression of p16(INK4A) and suppression of p53 and Rb proteins are key features of oncogenic transformation by human papillomaviruses (HPV) in anogenital cancers. HPV genomes are often detected in cancers of the oral cavity, but it is unclear whether HPV has a specific oncogenic role there.
The objectives of the study were to investigate the expression of p53, Rb, and p16(INK4A) proteins and identify HPV infection and viral integration into the host genome.
Seventy-nine cases of oral squamous cell carcinoma (OSCC) were studied by immunohistochemistry. Polymerase chain reaction (PCR) was performed to identify HPV DNA from the samples. The results were correlated with clinical data.
Thirty-three cases were HPV positive for high-risk HPV (HR-HPV) types, of which 27 harbored HPV16. In 25 of 27 HPV16-positive tumors, the HPV16 genome was fully integrated into the host genome, as evidenced by the lack of PCR-amplifiable E2 gene sequences. Forty-five patients were p53 overexpressing, 20 with HR-HPV-positive and 25 with HR-HPV-negative tumors. p16(INK4A) protein was overexpressed in 4 of 31 HR-HPV-positive and 9of 45 HR-HPV-negative cases. Twenty-six of 32 HR-HPV-positive and 37 of 44 HR-HPV-negative samples exhibited pRb nuclear staining. These differences between HR-HPV-positive and -negative tumors were not statistically significant. No correlation was found between these biological factors and tumor location, stage, differentiation grade, or alcohol or tobacco abuse.
A tumor immunophenotype, similar to HPV-related anogenital cancers, is not present in OSCC and highly oncogenic HPV types are therefore unlikely to be specific or independent risk factors for oral cancer.